Evaluación toxicológica in vitro e in vivo de compuestos organosulfurados con potencial uso en envasado activo en contacto con alimentos

UNIVERSIDAD DE SEVILLA FACULTAD DE FARMACIA DEPARTAMENTO DE NUTRICIÓN Y BROMATOLOGÍA, TOXICOLOGÍA Y MEDICINA LEGAL “EVALUACIÓN TOXICOLÓGICA IN VITRO E IN VIVO DE COMPUESTOS ORGANOSULFURADOS CON POTENCIAL USO EN ENVASADO ACTIVO EN CONTACTO CON ALIMENTOS” Memoria que presenta la Licenciada MARÍA PILAR MELLADO GARCÍA para optar al título de Doctor por la Universidad de Sevilla con la Mención Internacional Sevilla, 2016 UNIVERSIDAD DE SEVILLA AREA DE TOXICOLOGÍA C/ Profesor García González, nº 2 41012 Sevilla (España) Teléfono: 954 55 67 62 Fax: 954 55 64 22 Este trabajo ha sido realizado en el Área de Toxicología del Departamento de Nutrición y Bromatología, Toxicología y Medicinal Legal de la Facultad de Farmacia de la Universidad de Sevilla y financiado por los siguientes Proyectos de Investigación:  Contratada como “Personal Investigador” asociado al proyecto del Ministerio de Ciencia e Innovación: AGL2012-38357-C “Evaluación de la Seguridad para Contacto Alimentario de Envases Activos Basados en Polímeros Biodegradables y Extractos Naturales” en el Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal, Área de Toxicología, Facultad de Farmacia, Universidad de Sevilla. Desde Junio 2014 – diciembre 2015. (Investigadora Principal: Ana María Cameán Fernández).  Miembro del equipo de investigación del proyecto de excelencia P11- AGR-7252 de la Junta de Andalucía, denominado “Desarrollo y Evaluación toxicológica de envases activos aptos para contacto alimentario” (Investigadora Principal: Ana María Cameán Fernández).  V Plan Propio Ayuda suplementaria a grupos de investigación por el cumplimiento del contrato-programa entre la Ceice y la U.S, desde (15/03/2016 a 14/05/2016) (Investigadora Principal: Ana María Cameán Fernández).  Contratada como personal investigador con orgánica y número de proyecto: 18 10062903 2012/971 de la Universidad de Sevilla (15/11/2016 a 14/12/2016). I. Índice / Index Índice / Index ÍNDICE / INDEX I. RESUMEN/SUMMARY: ....................................................................................... 1 II. INTRODUCCIÓN/INTRODUCTION………………………………………………………………….……17 DEFINICIÓN Y TIPOS……………………………………………………….…………………………………....17 ABSORBEDORES O ELIMINADORES:…………………………………………………………..………………..……..21 LIBERADORES O EMISORES:……………………………………………………………………………………………..24 1.2 ASPECTOS LEGISLATIVOS EN ENVASES ACTIVOS ...................................................... 28 2.- ACEITES ESENCIALES Y SUS PROPIEDADES ................................................. 32 2.1 ACEITES ESENCIALES EN LA INDUSTRIA ALIMENTARIA, USO EN EL ENVASE ACTIVO ............ 33 2.2 ALIÁCEAS Y SUS PROPIEDADES .......................................................................... 37 2.3 COMPUESTOS ORGANOSULFURADOS PRESENTES EN ALIÁCEAS CON POTENCIAL USO EN LA INDUSTRIA ALIMENTARIA ...................................................................................... 39 2.4. PROPIL PROPANO TIOSULFINATO (PTS), PROPIL PROPANO TIOSULFONATO (PTSO). ..... 43 3. SEGURIDAD DE LOS ENVASES ACTIVOS. ESTUDIOS TOXICOLÓGICOS ........... 45 3.1 PANORAMA ACTUAL………………………………………………………………………………………………..45 3.2 ESTUDIOS DE CITOTOXICIDAD Y MUERTE CELULAR IN VITRO DE OS .............................. 55 3.3 ESTUDIOS DE GENOTOXICIDAD/MUTAGENICIDAD IN VITRO ...................................... 57 3.4. ESTUDIOS DE TOXICIDAD IN VIVO...................................................................... 61 III. JUSTIFICACIÓN Y OBJETIVOS / SIGNIFICANCE AND PURPOSES……..……………….………………65 IV. RESULTADOS Y DISCUSIÓN / RESULTS AND DISCUSSION……………..………………………………75 CAPÍTULO I / Chapter I. Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from allium extract, intended to food active packaging…………77 CAPÍTULO II / Chapter II. Genotoxicity of a thiosulfonate compound derived from allium sp. intended to be used in active food packaging: in vivo comet assay and micronucleus test……………………………………………………………………………………………………………………………....89 CAPÍTULO III / Chapter III. Toxicological evaluation of an allium-based commercial product in a 90-day feeding study in sprague–dawley rats…………………………………………..103 CAPÍTULO IV / Chapter IV. In vitro toxicological assessment of an organosulfur compound from allium extract: cytotoxicity, mutagenicity and genotoxicity study……………………….………119 CAPÍTULO V / Chapter V. Resultados pendientes de publicación como artículos / Results pending publication………………………………………………………………………………………….………………….131 V.I. Determinación de la mortalidad celular, estrés oxidativo y fagocitosis en macrófagos de células thp-1 mediante citometría de flujo………………………………………………………………………133 V. DISCUSIÓN GENERAL / GENERAL DISCUSSION………………………………………………………………159 VI. CONCLUSIONES / CONCLUSIONS………………………………………………………………………………….175 VII. REFERENCIAS / REFERENCES……………………………………………………………………………………….181 Resumen/Summary 1 En la actualidad, la industria alimentaria está apostando por la incorporación de sustancias naturales a envases alimentarios con el fin de incrementar la perdurabilidad de los alimentos en el mercado. Con estas prácticas se intentan satisfacer las necesidades de los consumidores de tal modo que su uso suponga ventajas tecnológicas y beneficios para el consumidor. Sin embargo, para que una sustancia sea admitida como aditivo debe estar bien caracterizada químicamente y debe superar los controles toxicológicos establecidos por parte de los correspondientes organismos sanitarios debido al desconocimiento del uso de estos componentes y sus posibles consecuencias. Entre las sustancias naturales que se emplean con este fin destacan los AEs, los cuales han sido tradicionalmente utilizados por sus propiedades farmacológicas. En nuestro caso, tanto un extracto del género Allium, denominado comercialmente PROALLIUM AP®, como alguno de sus componentes (PTSO y su análogo PTS) pretenden ser utilizados como antimicrobianos en la industria alimentaria formando parte de envases activos. Como paso previo al estudio de su seguridad se realizó una revisión bibliográfica de los datos de toxicidad disponibles hasta el momento en la literatura científica, tras la cual se puso de manifiesto la ausencia de estudios requeridos en diferentes compuestos con interés en la conservación de alimentos y la disparidad de los resultados disponibles. La evaluación toxicológica realizada en la presente tesis doctoral comenzó con una batería de ensayos tanto in vitro, con células que estarían en contacto con estas sustancias al ser ingeridas, como in vivo, en ratas tras un consumo agudo y crónico, para investigar los posibles efectos tóxicos, destacando los estudios de genotoxicidad, que pueden desencadenarse tras la exposición a PROALLIUM AP®, PTSO y PTS. La relevancia de la información toxicológica de estas sustancias resulta fundamental para su futuro uso ya que constituye un requisito reglamentario por parte de las autoridades competentes antes de su comercialización. Todos estos experimentos dieron lugar a las siguientes publicaciones:  GENOTOXICITY ASSESSMENT OF PROPYL THIOSULFINATE OXIDE, AN ORGANOSULFUR COMPOUND FROM ALLIUM EXTRACT, INTENDED TO FOOD Resumen/Summary 2 ACTIVE PACKAGING. (Mellado-García y cols., 2015), Food and Chemical Toxicology 86, 365-373. La vía de exposición más importante en el contexto que nos engloba es la vía oral ya que una vez el extracto de PROALLIUM AP® sea incorporado en films en envases activos, PTSO podría ser ingerido por los consumidores. En este trabajo, las células Caco-2 (adenocarcinoma de colon), fueron expuestas a diferentes concentraciones en función de la concentración máxima que migraría del film al consumidor en el peor escenario posible. Los experimentos llevados a cabo incluyen la evaluación de la mutagenicidad de PTSO en diferentes cepas de S. typhimurium (0-20 μM) cada una con características diferentes alteradas genéticamente para presentar mutaciones en los genes implicados en la síntesis de histidina para abarcar un amplio rango de posibles mutaciones. También se estudió la mutagenicidad en las células L5178Y TK+/- de mamíferos (ensayo de MLA), tras la realización de un estudio de citotoxicidad previo, en el que se determinaron las concentraciones de exposición a 4h (0-30 μM) y a 24h (0-20 μM). En el caso del test de Ames, no hubo diferencias significativas en ninguna de las cepas estudiadas en ausencia ni en presencia de S9. Sin embargo, a las 24 h de exposición en el ensayo de MLA se observaron diferencias significativas en el recuento de colonias en el rango de 2,5-20 μM. Por otro lado, PTSO no indujo incrementos en el porcentaje de MN (0-40 μM) en ausencia de S9 a ninguna de las concentraciones ensayadas, pero sí en presencia de la fracción microsómica S9 a partir de 15 μM, indicando la genotoxicidad de su metabolito. Por último, el ensayo cometa (0-50 μM) no mostró rotura ni daño oxidativo en el ADN de las células Caco-2 tratadas. Posteriormente, teniendo en cuenta los resultados contradictorios de genotoxicidad de PTSO in vitro, siguiendo las recomendaciones de la EFSA (EFSA 2011), se procedió al estudio de la genotoxicidad in vivo de PTSO en ratas Wistar, mediante el siguiente trabajo:  GENOTOXICITY OF A THIOSULFONATE COMPOUND DERIVED FROM ALLIUM sp. INTENDED TO BE USED IN ACTIVE FOOD PACKAGING: IN VIVO COMET ASSAY Resumen/Summary 3 AND MICRONUCLEUS TEST. (Mellado-García y cols., 2016), Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 800-801, 1-11. Se procedió al estudio de la genotoxicidad mediante el ensayo de MN en la sangre de la médula ósea de las ratas Wistar, así como el ensayo cometa en estómago e hígado de las mismas, por ser el primer órgano de contacto en la ingestión de PTSO y el principal órgano de metabolismo de xenobióticos, respectivamente. Este estudio fue realizado administrando a las ratas dosis de 55; 17,4; 5,5 mg/kg p.c. de PTSO. Tras la necropsia, se realizó un estudio histopatológico de ambos órganos, no observándose indicios de genotoxicidad al microscopio óptico ni al microscopio electrónico en las ratas expuestas respecto del control. Solamente, a la concentración más alta ensayada se observó un incremento del almacenamiento de glucógeno en hígado y procesos degenerativos en estómago con vacuolización de las membranas celulares. El estudio se completó con un análisis para determinar la presencia de PTSO mediante cromatografía UHPLC-MS/MS Orbitrap en la sangre de las ratas tratadas, no detectándose PTSO en ningún caso. Por último, para corroborar la ausencia de genotoxicidad en los órganos estudiados previamente, se decidió evaluar la presencia de PTSO en los tejidos empleando la técnica de pirólisis analítica mediante cromatografía gaseosa combinada con un detector de masas (Py-GC-MS). Se demostró la existencia en hígado de derivados del componente principal y dos posibles metabolitos, lo que confirmó el fenómeno de metabolismo de PTSO en el organismo. Tras el estudio de toxicidad aguda de PTSO en ratas, de nuevo siguiendo las recomendaciones de la EFSA (EFSA, 2011), con el fin de completar los resultados de genotoxicidad obtenidos, y dada la escasez de información in vivo, se realizó un ensayo de toxicidad crónica durante 90 días con PROALLIUM AP®.  TOXICOLOGICAL EVALUATION OF AN ALLIUM-BASED COMMERCIAL PRODUCT IN A 90-DAY FEEDING STUDY IN SPRAGUE-DAWLEY RATS. (Mellado-García y cols., 2015), Food and Chemical Toxicology 90, 18-29. Resumen/Summary 4 El PROALLIUM AP® es un extracto de diferentes componentes presentes en Allium sp. que será incorporado en los films anteriormente mencionados por su actividad antimicrobiana. En este sentido, tras la realización de los ensayos anteriores, debido a la falta de información de este componente y siguiendo las recomendaciones del Comité Científico en alimentación de la Unión Europea, el cual requiere la evaluación de las sustancias usadas en materiales en contacto con alimentos, se realizó un estudio de toxicidad subcrónica oral en ratas Sprague-Dawley por un periodo de exposición por vía oral de 90 días. Para ello se emplearon dosis de 0, 25, 100 y 400 mg/kg/día PROALLIUM AP®. Las ratas fueron sacrificadas y se extrajeron sus órganos (hígado, riñón, intestino, cerebro, timo, epidídimo, glándula adrenal, corazón, testículos/ovarios, pulmones y bazo) y sangre por punción cardíaca. Adicionalmente, las ratas fueron controladas cada semana midiendo el peso, el consumo de agua y comida, y se añadió un estudio histopatológico, bioquímico clínico y hematológico de las ratas expuestas. Las ratas no mostraron signos clínicos de mortalidad dosis-relacionados. Los resultados no mostraron diferencias significativas a ninguna de las concentraciones expuestas respecto del control, en ninguno de los parámetros estudiados. De esta forma, se determinó el NOAEL de PROALLIUM AP® en 400 mg/kg/día, un valor 500 veces superior al de la exposición derivada de su potencial uso en envase activo. Además con el fin de estudiar otro componente OS con potencial aplicación en la industria alimentaria, PTS, al cual se le atribuyen también propiedades antimicrobianas, se realizó la evaluación de la citotoxicidad, mutagenicidad y genotoxicidad in vitro del mismo, en la siguiente publicación:  IN VITRO TOXICOLOGICAL ASSESSMENT OF AN ORGANOSULFUR COMPOUND FROM ALLIUM EXTRACT: CYTOTOXICITY, MUTAGENICITY AND GENOTOXICITY STUDIES. (Mellado-García y cols., 2017), Food and Chemical Toxicology 90, 231–240. Resumen/Summary 5 El objetivo de este estudio fue la realización por primera vez del estudio de la citotoxicidad de PTS en células Caco-2 a 24 y a 48h, determinando su CE50 (280 μM) a través de distintos biomarcadores de viabilidad (RN, MTS, CP). Además se realizó una evaluación de la mutagenicidad en el test de Ames (0-280 μM), en el cual se utilizaron 5 cepas de S. typhimurium. En este ensayo, no se observaron diferencias significativas a ninguna de las concentraciones ensayadas, en presencia o en ausencia de S9. Por otro lado, en el ensayo de MLA, no se observaron diferencias significativas en el ensayo ni tras 4 ni 24h de exposición, demostrando así la ausencia de mutagenicidad. Además de estos ensayos, se realizó una evaluación de la genotoxicidad mediante el test de MN en las células L5178Y TK+/- tanto en ausencia (0-17,25 μM) como en presencia (0-25 μM) de S9. En este ensayo, se detectó un aumento de la frecuencia de células binucleadas con MN a la concentración más elevada ensayada sin S9 (17,25 μM), y a las dos concentraciones más altas con S9 (20-25 μM), mostrando que tanto los metabolitos como el componente original producen genotoxicidad. Por último, se estudió la genotoxicidad mediante el ensayo cometa estándar (0-280 μM) y modificado con enzimas de restricción en células Caco-2. En este caso, solamente se observaron daños en el ADN a la concentración más alta ensayada en el ensayo cometa estándar, mientras que no se observaron daños oxidativos en el ensayo cometa modificado a ninguna concentración. Para concluir, para la realización de esta tesis doctoral, la doctoranda realizó una estancia internacional en el departamento de “SEBIO, Stress Environnementaux et Biosurveillance de milieux aquatiques” en la Universidad de Reims, Champagne-Ardennes, (Francia). Esta estancia se realizó bajo la dirección del Doctor Stéphane Bettoulle, el Doctor Alain Geffard como director del departamento y con la colaboración y supervisión del doctorando Hakim Samai. Tras los resultados obtenidos previamente in vitro, sobre distintos tipos celulares, unidos a los ya realizados anteriormente por nuestro laboratorio sobre PTSO, se decidió aprovechar esta estancia internacional para estudiar la mortalidad celular de las células THP-1 (leucemia monocítica aguda), en estado macrófago, así como el estrés oxidativo y la fagocitosis en la exposición de diferentes concentraciones de PTSO Resumen/Summary 6 durante 24h mediante citometría de flujo. Los resultados obtenidos de este experimento dieron lugar a la siguiente publicación: • “DETERMINACIÓN DE LA MORTALIDAD CELULAR, ESTRÉS OXIDATIVO Y FAGOCITOSIS EN MACRÓFAGOS DE CÉLULAS THP-1 MEDIANTE CITOMETRÍA DE FLUJO”, Revista Española de Toxicología (pendiente de publicación). En este ensayo se estudió el comportamiento de PTSO en las células THP-1, células de leucemia monocítica humana midiendo la mortalidad celular, el estrés oxidativo y la fagocitosis mediante citometría de flujo. Para ello, las células THP-1 fueron activadas a estado macrófago, obteniéndose un aumento significativo de la mortalidad celular a partir de 60 μM de PTSO. Por otro lado, no se observaron aumentos significativos de la producción de especies reactivas de oxígeno a ninguna de las concentraciones de exposición. Por último, se estudió la fagocitosis utilizando microesferas fluorescentes de látex, que mostraron diferencias significativas a 60 μM de PTSO y a la concentración más alta ensayada (150 μM de PTSO). Además, se estudió la actividad fagocitaria de THP-1 dando como resultados diferencias significativas a 60 μM y a 150 μM. Por último, se determinó el número medio de microesferas fagocitadas por célula, obteniéndose diferencias significativas a las dos concentraciones más altas ensayadas respecto del control negativo (100 y 150 μM) siendo un total de 6 microesferas/célula. Teniendo en cuenta estos resultados, podríamos decir que las concentraciones que pueden llegar a producir efectos tóxicos in vitro son inferiores a las que se esperan puedan llegar al consumidor en el peor escenario posible (37.5 µM de PTSO). Resumen/Summary 7 Currently, the food industry is betting on the incorporation of natural substances into food packaging in order to increase the durability of food in the market. These practices are intended to satisfy the needs of consumers so that their use entails technological advantages and benefits for the consumer. However, in order to admit a substance as an additive, it must be well characterized chemically and must overcome the toxicological assessment established by the corresponding health agencies, due to the lack of knowledge of the use of these components and their possible consequences. Among the natural substances used for this purpose are Essential Oils (EOs), which have traditionally been used for their pharmacological properties. In our case, both an extract of the genus Allium, commercially known as PROALLIUM AP®, as well as some of its components (PTSO and its analog PTS) are intended to be used as antimicrobials in the food industry as part of active packaging. As a preliminary step to the study of its safety, a bibliographical review of the toxicity data available so far was made in the scientific literature. Afterwards, the lack of studies required in different compounds with interest in food preservation and the disparity of available results have been evidenced. The toxicological evaluation carried our in the present Thesis started with a battery of tests both in vitro, with cells that would be in contact with these substances when ingested, and in vivo, in rats after acute and chronic consumption, in order to investigate possible toxic effects, mainly genotoxicity, which may be triggered upon exposure to PROALLIUM AP®, PTSO and PTS. Tre relevance of the toxicological information of these substances is essential for their future use since it is a regulatory requirement by the competent authorities prior to their commercialization. All these experiments has led to the following publications:  GENOTOXICITY ASSESSMENT OF PROPYL THIOSULFINATE OXIDE, AN ORGANOSULFUR COMPOUND FROM ALLIUM EXTRACT, INTENDED TO FOOD ACTIVE PACKAGING. (Mellado-García y cols., 2015), Food and Chemical Toxicology 86, 365-373. Resumen/Summary 8 The most important route of exposure in our context is the oral route because once the PROALLIUM AP® extract is incorporated into films in active packaging, PTSO could be ingested by consumers. In this work, Caco-2 cells (colon adenocarcinoma) were exposed to different concentrations depending on the maximum concentration that would migrate from the film to the consumer in the worst possible scenario. Experiments carried out in our laboratory included the evaluation of the mutagenicity of PTSO in different strains of S. typhimurium (0-20 μM) each one with different genetically altered characteristics to present mutations in genes involved in histidine synthesis to cover a wide range of possible mutations. Mutagenicity in mammalian L5178Y TK +/- cells (MLA assay) was also studied following a previous cytotoxicity study, in which the exposure concentrations were determined at 4h (0-30 μM) and at 24h ( 0-20 μM). In the case of the Ames test, there were no significant differences in any of the strains studied in the absence or presence of S9. However, at 24 h of exposure in the MLA assay, significant differences were observed in the revertant colonies in the range of 2.5-20 μM. On the other hand, PTSO did not induce increases in the percentage of MN (0-40 μM) in the absence of S9 at any of the concentrations tested, but it did in the presence of the microsomal fraction S9 from 15 μM, indicating the genotoxicity of its metabolite. Finally, the comet assay (0-50 μM) showed that PTSO did not induce DNA strand breaks or oxidative damage in the DNA of Caco-2 cells exposed. Afterwards, considering the contradictory results of PTSO obtained in genotoxicity in vitro, following the recommendations of the EFSA (EFSA 2011), the in vivo genotoxicity of PTSO in Wistar rats was studied in the following work:  GENOTOXICITY OF A THIOSULFONATE COMPOUND DERIVED FROM ALLIUM sp. INTENDED TO BE USED IN ACTIVE FOOD PACKAGING: IN VIVO COMET ASSAY AND MICRONUCLEUS TEST. (Mellado-García y cols., 2016), Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 800-801, 1-11. Resumen/Summary 9 Genotoxicity was studied by the MN test in the bone marrow in Wistar rats, and with the comet assay in stomach and liver of rats, being the first contact organ in the ingestion of PTSO and the main metabolism organ of xenobiotics, respectively. This study was performed by administering to the rats doses of 55; 17.4; 5.5 mg/kg b.w. of PTSO. After necropsy, a histopathological study of both organs was performed, with no evidence of genotoxicity under optical microscopy or electron microscopy in rats exposed to the control. Only at the highest concentration tested an increase in glycogen storage in liver and degenerative processes in the stomach with vacuolization of cell membranes were observed. The study was completed with an analysis to determine the presence of PTSO by UHPLC-MS/MS Orbitrap chromatography in the blood of treated rats, with no PTSO detected. Finally, in order to corroborate the absence of genotoxicity in the organs previously studied, the evaluation of the presence of PTSO in the tissues using the analytical pyrolysis technique by gas chromatography combined with a mass detector (Py-GC-MS) was carried out. It was demonstrated the existence in liver of derivatives of the main component and two possible metabolites, which confirmed the phenomenon of metabolism of PTSO in the organism. Following the acute toxicity study of PTSO in rats, and again following the recommendations of the EFSA (EFSA, 2011), in order to complete the results of genotoxicity obtained, and given the lack of in vivo information, a test of chronic toxicity for 90 days with PROALLIUM AP® was carried out.  TOXICOLOGICAL EVALUATION OF AN ALLIUM-BASED COMMERCIAL PRODUCT IN A 90-DAY FEEDING STUDY IN SPRAGUE-DAWLEY RATS. (Mellado-García y cols., 2015), Food and Chemical Toxicology 90, 18-29. PROALLIUM AP® is an extract of different components present in Allium sp. This extract will be incorporated into the films mentioned above for their antimicrobial activity. In this regard, following the previous tests, due to the lack of information on this component and following the recommendations of the Scientific Committee on Food of the European Union, which requires the evaluation of substances used in food contact materials. An oral subchronic toxicity study was performed on Sprague-Dawley Resumen/Summary 10 rats for a 90-day oral exposure period. The doses: 0, 25, 100 and 400 mg/kg/day PROALLIUM AP® were used. Rats were sacrificed and their organs (liver, kidney, intestine, brain, thymus, epididymis, adrenal gland, heart, testicles/ovaries, lungs and spleen) and blood by cardiac puncture were extracted. In addition, rats were monitored weekly for weight, water and food consumption, and a histopathological, clinical and hematological study of the exposed rats was added. Rats showed no clinical signs of dose-related mortality. The results did not show significant differences at any of the concentrations exposed to the control, in any of the parameters studied. In this way, PROALLIUM AP® NOAEL was determined at 400 mg/kg/day, a value 500 times higher than exposure derived from its potential use in active packaging. In addition, in order to study another OS component with potential application in the food industry, PTS, to which antimicrobial properties are also attributed, its in vitro cytotoxicity, mutagenicity and genotoxicity evaluation were performed in the following publication:  IN VITRO TOXICOLOGICAL ASSESSMENT OF AN ORGANOSULFUR COMPOUND FROM ALLIUM EXTRACT: CYTOTOXICITY, MUTAGENICITY AND GENOTOXICITY STUDIES. (Mellado-García y cols., 2016), Food and Chemical Toxicology (in press) The objective of this study was to study for the first of cytotoxicity of PTS in Caco-2 cells at 24 and 48h, determining its CE50 (280 μM) through different biomarkers of viability (RN, MTS, CP). Moreover, an evaluation of the mutagenicity in the Ames test (0-280 μM) was carried out, in which 5 strains of S. typhimurium were used. In this test, no significant differences were observed at any of the concentrations tested, in the presence or absence of S9. On the other hand, in the MLA assay, no significant differences were observed in the assay either after 4 or 24 hours of exposure, thus demonstrating the absence of mutagenicity. In addition to these assays, an assessment of genotoxicity was performed by the MN test on L5178Y TK+/- cells both in the absence Introducción/Introduction 19 ejemplo, entrada de aire en un producto envasado al vacío o fuga de gases en envases con atmósfera protectora (AECOSAN, 2010). Dentro de este tipo, se encuentran los Indicadores de Tiempo-Temperatura (ITT), indicadores de madurez, detectores de sustancias volátiles de origen microbiano: dióxido de carbono (CO2), compuestos nitrogenados, detectores de toxinas e incluso bacterias patógenas en sí mismas, controladores de la temperatura e identificadores de radiofrecuencia (mecanismo ideal para el seguimiento y localización de alimentos perecederos). Estos indicadores pueden encontrarse en el interior del envase o adheridos en el exterior del mismo (Brody y cols., 2008; Dainelli y cols., 2008). Centrándonos en el concepto de envases activos, existen diferentes sistemas que se clasifican según su mecanismo de acción (Tabla 1): Estos envases incluyen sistemas que absorben/eliminan o regulan compuestos que afectan desfavorablemente a la vida comercial de un producto alimenticio, tales como el oxígeno, el etileno, la humedad o aquellos que pueden ocasionar olores o sabores desagradables en los alimentos. Por otro lado, otros sistemas liberan sustancias químicas como conservantes, antioxidantes, colorantes, aromas, etc. (AECOSAN, 2010). La siguiente generación incluye materiales con propiedades antimicrobianas. Esta tecnología dentro de los envases, juega un papel crucial extendiendo la vida útil de los alimentos y reduciendo el riesgo de contaminación por patógenos (Appendini y Hotchkiss, 2002). 1.- Absorbedores o eliminadores 2.- Liberadores o emisores a) Absorbedores de oxígeno a) Liberadores de Etanol b) Absorbedores de dióxido de carbono b) Liberadores de dióxido de carbono c) Absorbentes de la humedad c) Liberadores de aromas, colorantes y otros ingredientes alimentarios d) Absorbentes de etileno d) Liberadores de antioxidantes y/o conservantes e) Absorbedores de olores/sabores indeseables e) Liberadores de extractos naturales: sustancias antimicrobianas/ biocidas Tabla 1: Clasificación de materiales activos, tomada de Chaves (2011). Introducción/Introduction 20 La catalogación de un material como activo, se basa en las propiedades del polímero utilizado como material de envasado, o en la inclusión de determinadas sustancias, ya sea en su superficie, en estructuras multicapa o en elementos asociados al envase, tales como bolsitas, láminas, etiquetas o tapones de botellas (Chaves, 2011). La tecnología del envase activo modifica determinadas condiciones o procesos del alimento que juegan un papel crucial en la vida útil del mismo, por ejemplo procesos químicos (oxidación, decoloración), físicos (endurecimiento o deshidratación) o microbiológicos (deterioro por microorganismos) (AECOSAN, 2010). Sería por tanto posible, a través de por ejemplo: el recubrimiento, el microperforado, la laminación, la coextrusión, o mezcla de polímeros, los alimentos podrían ser manipulados modificando la permeabilidad/selectividad atmosférica de compuestos gaseosos dentro de un envase, con respecto a la oxidación o la respiración cinética de alimentos. Por otro lado, ciertos materiales nanocompuestos sirven también como envases activos previniendo la llegada del O2, CO2, y la humedad a los alimentos (Brody y cols., 2008). Además, existen nanomateriales cuya principal función es la de mejorar las propiedades mecánicas y de barrera de los materiales de envasado (Henriette, 2009). Tal y como se ha citado anteriormente, se pueden distinguir atendiendo a su mecanismo de acción en dos grandes grupos:  Absorbedores o eliminadores: son aquellos que absorben sustancias químicas del interior del envase. Su acción implica una transferencia de masa desde el contenido del envase al sistema activo.  Liberadores o emisores: liberan sustancias químicas al interior del envase. Su acción implica una transferencia de masa desde el sistema o material activo al contenido del envase (AECOSAN, 2010). Comercialmente se pueden encontrar como:  Sistemas independientes: saquitos, tiras o etiquetas, constituyendo un elemento que se incorpora al interior del envase, realizados con materiales Introducción/Introduction 21 resistentes y estratégicamente colocados hacia el espacio de cabeza y no en contacto con los alimentos. Deben diferenciarse claramente del envase y del contenido, acompañados de un etiquetado apropiado que permita al consumidor su identificación y evite su consumo accidental (Ozdemir y Floros, 2004).  Sistemas integrados en el propio envase: Las sustancias activas se incorporan en el propio material de envase, en unos casos mediante dispersión y en otros insertándolas químicamente en el propio material. Se puede incorporar en capas intermedias de los materiales multicapa, evitando así el contacto directo de la parte activa con el alimento o bien directamente en contacto con el alimento. En este caso, este sistema es indistinguible del resto del envase por parte del consumidor, evita su rechazo o su ingestión accidental (AECOSAN, 2010). Absorbedores o eliminadores:  Absorbedores de O2: en muchos casos, el deterioro de los alimentos es debido a la oxidación de sus constituyentes o el deterioro por microorganismos en presencia de O2 (De Krujif y cols., 2002). El funcionamiento de estos absorbedores se basa en la utilización de diversos mecanismos solos o combinados (Suppakuk y cols., 2003; Rooney, 2005): oxidación de hierro y sales ferrosas, oxidación de sustancias orgánicas tales como el ácido ascórbico, catecol, ácidos grasos insaturados, y oxidación enzimática. Los absorbedores reducen y controlan niveles inferiores al 0,01% de oxígeno (AESAN, 2010). Ejemplos de principios de este tipo de tecnología, incluyen hierro o complejos de hierro, película de nylon con un catalizador de cobalto, ácido ascórbico, sales de sulfito y las enzimas como la glucosa oxidasa y la alcohol oxidasa (Informe PACMan, 2013). Se utilizan en productos alimenticios sensibles al O2: frutos secos, pan y productos de bollería, pastas, pizzas, chocolates y bombones, cerveza, zumos, etc. Dentro de este grupo encontramos los absorbedores de radicales libres de O2. Se basa en el hecho de que el proceso de oxidación tiene lugar a través de una reacción en cadena iniciada por radicales libres generados a partir del O2. Así, si en lugar de Introducción/Introduction 22 eliminarlo se eliminan los radicales libres a medida que se producen, la oxidación no tendría lugar (AECOSAN, 2010). Aceites esenciales y componentes fenólicos se incorporan en materiales de envase con la finalidad de reaccionar con radicales libres de O2 para eliminarlos del espacio de cabeza, ralentizar los procesos de oxidación e incrementar la vida comercial del producto (Nerín y cols., 2008; Pezo y cols., 2008; Nerín, 2010). Absorbedores de CO2: El CO2 se forma en algunos alimentos debido al deterioro y las reacciones de respiración, por lo que debe ser eliminado del envase por captadores de CO2 para evitar el deterioro del alimento y del propio envase, como ocurre con el café tostado que produce volúmenes significativos de este gas (Vermeiren y cols., 1999). Saquitos conteniendo óxido de calcio y un agente higroscópico se utilizan para retener como carbonato el CO2 formado y eliminarlo del espacio de cabeza (Ozdemir y cols., 2004). Absorbedores de humedad: El exceso de humedad es la causa principal del deterioro de los alimentos, debido a la posibilidad de proliferación de los microorganismos, así como una disminución del atractivo por parte del consumidor (Chaves, 2011). Los productos envasados con un alto valor de humedad relativa en el espacio de cabeza, son susceptibles a las fluctuaciones de temperatura durante el transporte o almacenamiento, lo que favorece la formación de condensados y nieblas (AESAN, 2010). Los captadores o reguladores de humedad (saquitos higroscópicos) o polímeros superabsorbentes localizados entre dos capas de polímeros con microporos, tienen como finalidad reducir la actividad del agua de los alimentos envasados (Vermeiren y cols., 1999). Se pueden utilizar fibras de celulosa, sales de poliacrilato, polipropilenglicol, carbohidratos, sales minerales, silicagel, montmorillonita, etc. Se utilizan en pescados, carnes, quesos, frutos secos, especias, productos de panadería, alimentos secos, frutas y vegetales cortados (AECOSAN, 2010). Introducción/Introduction 23 Absorbedores de etileno: La mayoría de las frutas y verduras liberan gas etileno después de la recolección (AECOSAN, 2010). El etileno actúa como una fitohormona y está relacionada con el proceso de deterioro de los alimentos, como la aceleración de la respiración, maduración, senescencia y ablandamiento de la fruta y verdura. Es por ello que es precisa la eliminación del etileno del envase para aumentar la vida útil de los alimentos y mantener las características organolépticas de los mismos (Chaves, 2011). Los métodos para eliminar el etileno se basan en la combinación de compuestos capaces de degradarlo, solos o acompañados de materiales adsorbentes (Fernández Alvarez, 2000). El principal componente activo para esta función en el mercado es el permanganato potásico que oxida el etileno hasta CO2 y agua (Vermeiren y cols, 1999). Además se utilizan otros sistemas basados en la capacidad que tienen determinados materiales de absorber el etileno, por ejemplo, minerales finamente divididos y dispersados como zeolitas, silicatos, carbón activo, etc. Se utiliza en frutas y verduras sensibles al etileno como: manzanas, kiwis, plátanos, mango, tomates y cebollas (AECOSAN, 2010). Absorbedores de olores/sabores indeseables: Estos componentes pueden tener orígenes diversos, como por ejemplo la oxidación de las grasas y aceites, la degradación de proteínas musculares en aminas y la formación de aldehídos, entre otros (Chaves, 2011). Además, durante la distribución y almacenamiento de muchos alimentos se producen reacciones comunes de deterioro, como la oxidación lipídica o glucolisis anaerobia que contribuyen a la formación de estos olores (López-rubio y cols., 2004). Los aldehídos pueden ser eliminados selectivamente, utilizando polímeros absorbentes modificados con otros materiales. No obstante, la eliminación de estas sustancias no está permitida cuando son indicadores del deterioro de un alimento, pues enmascararían al mismo e inducirían a error al consumidor (AECOSAN, 2010) Introducción/Introduction 24 Liberadores o emisores: Liberadores de etanol: Esta técnica es utilizada en spray sobre la superficie de productos de repostería para suprimir el crecimiento de mohos, bacterias y microorganismos (Ozdemir y Floros, 2004). Estas presentaciones se caracterizan por la inclusión de bolsitas liberadoras de etanol encapsulado sobre un material de soporte inerte, por ejemplo sílica, finamente dividido y con un grado de humedad adecuado, que es liberado al espacio de cabeza de los envases manteniendo así el efecto conservador con el fin de inhibir el crecimiento microbiano. Además de en productos de panadería y pastelería, se emplean en alimentos secos o semisecos (AESAN, 2010). Liberadores de CO2: Las funciones de los liberadores de CO2 en un envase es la de suprimir el crecimiento microbiano, por lo que son utilizados frecuentemente como un complemento a los captadores de O2 (Kerry y cols., 2006). La presencia de CO2 en el espacio de cabeza a concentraciones en el rango 10%-80% inhibe el crecimiento microbiano en los alimentos (AESAN, 2010). Así mismo, cuando se utilizan conjuntamente con sistemas captadores de O2 o en caso de atmósferas con CO2, cuando éste se disuelve parcialmente en el alimento, se puede originar un vacío parcial en el envase terminando con un colapso en caso de los envases flexibles (Kerry y cols., 2006). La utilización de estos sistemas está extendida en el envasado de productos como la carne roja, de ave, pescados y quesos para inhibir el crecimiento microbiano en la superficie y aumentar la vida comercial (Chaves, 2011). Liberadores de aromas, colorantes y otros ingredientes alimentarios: Se liberan sustancias que mejoran la deseabilidad del producto en el material plástico mejorando o realzando el aroma propio del producto fresco, o en otros casos Introducción/Introduction 25 valiéndose de la actividad liberadora del envase activo al abrir el envase (Gutiérrez y cols., 2010; López-Rubio y cols., 2006). Se utiliza en productos de envasado de zumos de frutas, productos con alto contenido en proteínas y grasas (Fundación EOI, 2015). Liberadores de antioxidantes y conservantes: Los antioxidantes se utilizan ampliamente como aditivos alimentarios para mejorar la estabilidad oxidativa de lípidos, y prolongar la vida útil, principalmente de productos secos y sensibles al O2. Si bien, también pueden incorporarse en los materiales de envase utilizando sistemas que liberan agentes antioxidantes al alimento y/o al espacio de cabeza, donde ejercen su acción, generalmente como eliminadores de radicales libres para ralentizar los procesos de oxidación que afectan a la calidad de los alimentos (Fundación EOI, 2015). Se ha demostrado la efectividad de la utilización tanto de antioxidantes sintéticos como la terbutilhidroquinona (TBHQ), butil hidroxianisol (BHA) y butil hidroxitolueno (BHT), como de antioxidantes naturales como la vitamina E, el ácido ascórbico y los extractos naturales ricos en compuestos fenólicos (tocoferol) y/o terpénicos, (Hotchkiss, 1997; Ozdemir y Floros, 2004; Nerín y cols., 2006; Tovar y col., 2005). Por otro lado, existe cierta preocupación relativa a los efectos fisiológicos tras consumir BHT, debido a su tendencia a acumularse en el tejido adiposo humano. Por tanto, sería conveniente buscar otros antioxidantes naturales, y que no supongan riesgos para la salud, como los aceites esenciales (AEs) de los cuales hablaremos a continuación. Como conservantes, hay muchas aplicaciones de films y revestimientos comestibles adicionados de sustancias autorizadas como aditivos conservantes, principalmente ácidos orgánicos, como el ácido ascórbico, sorbato potásico, ácido benzoico, propiónico (Coma, 2008; Han y Floros, 1997; Weng y Chen, 1997; Outtara y cols., 2000), etil p-hidroxibenzoato y metil p-hidroxibenzoato (Dobias y cols., 2000), nisina, pediocina, natamicina (Natrajan y Sheldon, 2000; Coma, 2008) y dióxido de azufre (Ozdemir y Floros, 2004). Introducción/Introduction 26 Liberadores de sustancias antimicrobianas/biocidas: La contaminación y el crecimiento microbiano reducen la vida comercial de los alimentos e incrementan el riesgo de enfermedades de origen alimentario, por lo que los materiales activos se convierten en una prometedora forma de este tipo de envases, (Chaves, 2011) en los que se centrará esta tesis doctoral. Este tipo de envases está diseñado para modificar las condiciones ambientales en contacto con el alimento con el fin de inhibir el crecimiento microbiano aumentando de esta forma la vida útil de los alimentos (López-Rubio y cols., 2004). Por sus propiedades antioxidantes y antimicrobianas se emplean diferentes productos naturales como aceites esenciales, extractos de plantas o componentes obtenidos de los mismos y otros productos también de origen natural como el própoleo (AESAN, 2010). Por ejemplo, se utilizan extractos como el etanólico de clavo (Hong y cols., 2000), de semillas de uva (Coma, 2008) y de orégano o romero (Camo y cols., 2008); aceites esenciales de orégano (Skandamis y Nychass, 2002), canela y canela enriquecida con cinamaldehído (Rodríguez y cols., 2007, 2008; Gutiérrez y cols., 2009), clavo (López y cols., 2006) y principios activos como alilisotiocianato (Shin y cols., 2010) (Vega-Lugo y Loong-Tak, 2009). Se utilizan en algunos alimentos como la carne fresca, quesos, pescados, frutas y verduras preparadas para el consumo en los que las técnicas convencionales de conservación frente al desarrollo de patógenos no es posible (tratamiento térmico, secado, irradiación, salado). Existen varios tipos: o El envase incorpora agentes antimicrobianos, saquitos o almohadillas volátiles, diseñados para liberarlos al espacio de cabeza del envase o directamente en el alimento para evitar la contaminación de microorganismos (Appendini y Hotchkiss, 2002; López-Rubio y cols, 2004; Sung y cols., 2013). o Incorporación de agentes antimicrobianos volátiles o no volátiles directamente en el polímero. o Revestimientos o adsorción de agentes antimicrobianos en la superficie de los polímeros. Introducción/Introduction 27 o Inmovilización de agentes antimicrobianos en polímeros mediante uniones iónicas o covalentes. o Uso de polímeros que son antimicrobianos per se (Chaves y cols, 2011). En el grupo de los biocidas, se encuentran el triclosan, que se utiliza fundamentalmente en películas de polietileno, demostrándose su eficacia en bacterias como Escherichia coli y Staphylococcus aureus (Camilloto y cols., 2009). El dióxido de cloro, es eficaz contra bacterias, hongos y virus, se libera sostenida y controladamente proporcionando un amplio espectro y sin dejar residuos en el alimento (Chaves, 2011). La nisina es la única bacteriocina aprobada como aditivo alimentario (en Estados Unidos y Australia) para retrasar el crecimiento de microorganismos alterantes y patógenos en productos cárnicos (Marcos y cols., 2013; Millette y cols., 2007; Nguyen y cols., 2008). Otros como la natamicina, aditivo alimentario (E 235), producida mediante un cultivo puro de bacterias Streptomyces natalensis, se utiliza predominantemente para la conservación y tratamiento antifúngico externo de productos lácteos, queso y salchichas (Streekstra y cols., 2016). Para concluir, existen además otro tipo de envases activos, aquellos materiales que modifican las propiedades físicas. Estos materiales por reacciones químicas: exotérmicas o endotérmicas producen el calentamiento o enfriamiento de los envases (Fernández Álvarez, 2000). Se utilizan materiales conocidos como susceptores, son aleaciones de materiales magnéticos (aluminio, hierro, cobre…) insertadas en un envase o en envases diseñados para ser utilizados en el microondas, estos transforman la energía microondas en energía infrarroja la cual es transferida al alimento en forma de calor, ayudando al cocinado de los alimentos produciendo y concentrando la energía en la superficie de los mismos (Wilson, 2007). También existen otros susceptores que contribuyen a la reacción de Maillard y la formación de corteza superficial en los alimentos (Chaves, 2011). Por último comentar que las combinaciones de los distintos materiales activos anteriormente son frecuentes. En la figura 1 se muestra un ejemplo comercializado en Estados Unidos: Introducción/Introduction 28 Figura 1: Combinación de distintas técnicas en envasado activo comercializado, Ultrazap, Paper Pak Industries, Estados Unidos (http://zapsoaker.com/food/ultrazap_xtendapak_tomatoes.php) 1.2 Aspectos legislativos en envases activos La legislación referente a envases varía significativamente de un país a otro. Habría que destacar que la comercialización de estos envases activos se centra en Estados Unidos, Japón y Australia. En Europa, debido tanto a la existencia de leyes más restrictivas como a los recelos de la industria alimentaria, los costes de inversión en investigación y desarrollo, unido a las costumbres de los consumidores, más conservadores en sus hábitos de consumo y más reticentes a los cambios, dificultan la salida al mercado de esta tecnología (Danielli y cols., 2008). Así, se deben tener en cuenta las siguientes consideraciones según el Reglamento Marco (CE) 1935/2004 que hace referencia a todos los materiales en contacto con alimentos. Únicamente los componentes incluidos en la lista comunitaria de sustancias autorizadas podrán utilizarse en componentes de los materiales y objetos activos e inteligentes. Para incluir una sustancia en esta lista comunitaria las solicitudes de autorización de sustancias constituyentes de materiales y objetos activos e inteligentes se presentarán ante la autoridad competente de un Estado miembro. En España, es la Agencia Española de Consumo y Seguridad Alimentaria y Nutrición (AECOSAN)-Ministerio Introducción/Introduction 35 investigaciones para desarrollar sistemas de liberación controlada de AEs, debido al riesgo asociado a algunos conservantes sintéticos (Tongnuanchan y Benjakul, 2014). Así mismo, es interesante considerar el impacto ambiental del envasado, por esto diversos trabajos se han centrado en el desarrollo de láminas plásticas con AEs inmersos (Vargas, y cols., 2008). Los AEs y sus componentes mayoritarios varían en olor y sabor, los cuales están clasificados según los tipos y cantidad de sus constituyentes (Tongnuanchan y Benjakul, 2014). Algunos AEs más frecuentemente estudiados con fines alimentarios son el orégano (Origanum vulgare), el romero (Rosmarinus officinalis), el té verde (Camellia sinensis), la canela (Cinnamomum zeylanicum), y el clavo (Eugenia caryophyllata) por sus actividades antimicrobianas, antibacterianas, antifúngicas y antioxidantes (Llana-Ruiz-Cabello y cols., 2015c). El timol, un análogo del carvacrol, ha sido incorporado por disolución directa en films aislados de proteínas de soja como envases de aceite de oliva (Hu y cols., 2012). Ambos, carvacrol y timol, son adecuados para su uso en microencapsulados en una matriz de polímero para la conservación de alimentos frescos (Guarda y cols., 2011). Cabe destacar, que dependiendo de su estructura, estos componentes tendrán un mecanismo de acción diferente, en el cual estará basada su actividad (Maisanaba y cols., 2016). Otro componente como el linalool, es el principal constituyente de diversos aceites esenciales (69,2 a 2,91%), como la albahaca, el cilantro, el té verde, la lavanda y el orégano, tiene un uso potencial en las películas a base de polietileno de baja densidad, con su componente antimicrobiano que puede mejorar la calidad y seguridad de los quesos. Además, es estable a altas temperaturas relativas, por ello, se podría utilizar en polímeros y ser usado en envases antimicrobianos (Suppakul et al., 2008). El AE de canela, (Cinnamomun zeylanicum) y el cinamaldehído (con una concentración del 85% en el AE de canela), presenta un alto potencial antioxidante, antibacteriano y antiinflamatorio, y es usado ampliamente en la industria alimentaria (Xing y cols., 2014). El AE de canela y la canela reforzada con cinamaldehído, han sido incorporadas en polipropileno y polietileno/etileno vinil acetato, copolímeros usados en la industria Introducción/Introduction 36 panadera (López y cols., 2007) o incluso en envases de papel en fresas (Rodríguez y cols., 2007) y tomates cherry (Rodríguez-Lafuente y cols., 2010). El Eugenol, es un importante antioxidante que ha demostrado tener propiedades antimicrobianas y una potencial aplicación en disoluciones directas en películas de embalaje o recubrimiento, tales como películas de metil celulosa (Sanla-Ead y cols., 2012). En muchos casos, los AEs tienen mayores efectos beneficiosos que los componentes sintetizados puros. Esto puede ser debido a la acción sinérgica de la mezcla de componentes que pueden actuar en distintas dianas moleculares. Sin embargo, algunos constituyentes pueden afectar a la tasa de absorción o a la biodisponibilidad de otros componentes (Seow y cols., 2014). En la siguiente tabla (Tabla 2) se muestran las especies de plantas que contienen componentes mayoritarios con aplicaciones en envases activos, revisado por Llana-Ruiz-Cabello y cols., (2015c). Componentes mayoritarios en AEs Planta Especie Partes de la planta Componentes principales (aproximadamente %)a Referencias Ajo Allium sativum Bulbos 2-Propenil tioacetonitrilo (43.25), trisulfuro metil 2-propenil (23.40), disulfuro di-2-propenil (20.87) Kirkpinar y cols., 2011 Albahaca Ocimum basilicum Hojas Linalool (69.2),α-Cadinol (2.56), γ-Cadineno (2.5), estragol (2.4), δ-Guaieno (2.10) Stajkovic y cols., 2007 and Beric y cols., 2008 Canela Cinnamomum zeylanicum Corteza (E)- Cinamaldehído (68.95) Unlu y cols., 2010 Cebolla Allium cepa Bulbos Propionaldehído (78.41–13.56), dipropildisulfuro (78.7–10.6), 3,4-dimetiltiofeno(13.23–0.35) Colina-Coca y cols., 2013 Cilantro Coriandrum sativum Hojas Linalool (26), E-2-decanol (20) Delaquis y cols., 2002 Citronella Cymbopogon winterianus Hojas Citronelal (20–30), Citronelol (10–15) Sinha y cols., 2014 Clavo Eugenia caryophyllata Flores Eugenol (58.2–48.82), cariofileno (36.94–13.99), eugenol acetato (22.34–3.89) Guan y cols., 2007 Hierba del limón Cymbopogon citratus Hojas Citral (50–88) Sinha y cols., 2014 Mejorana Origanum majorana Inflorescencias Apigenina (35.23), metil rosmarenato (31.58), ácido rosmarínico (15.15) Roby y cols., 2013 Menta poleo Mentha pulegium Hojas and inflorescencias Mentona (35.90), neomentol (9.20), 8-Hidroxi- δ- 4(5)-p-menten-3-ona (2.10) Teixeira y cols., 2012 Té verde Camellia sinensis Hojas Linalool (56.73), α-terpineol (9.37), p-cresol, 2,6- di-tert-butil (11.63), nonanaldehído (5.00), Tontul y cols., 2013 Introducción/Introduction 37 hexanal (4.49), 3-metilbutanal (3.41) Orégano Origanum compactum Hojas γ-Terpineno (22.90),carvacrol (22.0), timol (19.36), p-Cimeno (13.26), β-cariofileno (3.47), αterpineno (3.26), linalool (2.91), mirceno (2.58), α-Tuyeno (1.51), α-Pineno (1.15) Mezzoug y cols., 2007 Origanum onites Carvacrol (64.30), linalool (13.80), p-cymeno (7.10), γ-terpineno (3.50), mirceno (1.6), timol (1.4), α-terpineno (1.4), α-pineno (1.0) Bostancioglu y cols., 2012 Linalool (50.53), carvacrol (24.52), timol (15.66) Özkan y Erdogan, 2011 Palmarosa Cymbopogon martini Hojas Geraniol (65–85), geranil acetato (5–20) Sinha y cols., 2014 Romero Rosmarinus officinalis Hojas and cáliz Verbenona (21.76), camfor (14.6), safrola (12.3), borneol (10.4), 1,8-cineola (7.26), α-pineno (6.65), β-cariofileno (6.17), geraniol (5.75), βselineno (3.61), α-selineno (3.57) Sacchetti y cols., 2005 Timo Artemisia campestris Hojas β-pineno (41.0), p-cimeno (9.9), α-terpineno (7.9), limoneno (6.5), mirceno (4.1) β-felandreno (3.4), α-Pineno(3.2) Neffati y cols., 2008 Thymus vulgaris L. ácido cinámico (28.54), apigenina (8.88), luteolin- 7-o-rutinosa (7.65) Roby y cols., 2013 Tabla 2. Especies de plantas que contienen componentes mayoritarios con aplicaciones en envases activos, incluyendo su composición y sus respectivos AEs (porcentajes). Tomada de Llana-Ruiz-Cabello y cols. (2015c). Los AEs de ajo, orégano y tomillo y sus componentes (carvacrol y timol) han demostrado ser eficaces para retrasar el crecimiento de microorganismos alterantes en diversos alimentos (Cerisuelo y cols., 2013;. Emiroglu y cols., 2010; Galotto y cols., 2012; Jouki y cols., 2014; Sung y cols., 2014). Otros investigadores han centrado sus esfuerzos en la inhibición de patógenos como E. coli O157: H7 y Listeria monocytogenes con AE de clavo o de ajo incorporados en la extrusión del material (Song y cols., 2014; Souza y cols., 2011). También se ha desarrollado un film celulósico que contiene isotiocianato de alilo encapsulado en nanotubos de carbono que es capaz de reducir la carga microbiana en carne de pollo (Dias y cols., 2013). 2.2 Aliáceas y sus propiedades Las Aliáceas son una familia de plantas monocotiledóneas dentro del orden Liliales, con unos 30 géneros y más de 670 especies. Son plantas de bulbos o tallos subterráneos, con hojas largas, delgadas y flores en umbela. Algunos autores no reconocen esta familia, y agrupan a sus especies dentro de las Liliáceas (Liliaceae). Las Introducción/Introduction 38 especies que pertenecen a la familia de Allium se han utilizado durante mucho tiempo como un remedio para la prevención y tratamiento de ciertas enfermedades (Bagheri y cols., 2011). Los adjetivos asociados a estas plantas e incluso el nombre latino de Allium, derivado del celta "todos" significa picante, refleja la presencia de ciertos sabores y aromas, los cuales comparten un solo elemento: el azufre (Ray y cols., 2011). Muchos de estos compuestos de azufre contienen el grupo alilo, nombre derivado de Allium. La sola presencia de estos compuestos orgánicos de azufre define el carácter de esta especie. El amplio espectro de acciones terapéuticas se ha atribuido a estas sustancias (Ray y cols., 2011). Los compuestos activos de la familia Alliaceae tienen una actividad fisiológica muy amplia, y estructuralmente son compuestos orgánicos de azufre intermedios con enlaces inusuales (Stajner y cols., 2008; Sapunjieva y cols., 2012). El género Allium posee componentes conocidos por sus actividades como antibacterianos y antioxidantes (Benkeblia, 2004; Ye y cols, 2013), además, de antimutagénicos, antiasmáticos, inmunomoduladores y prebióticos (Corzo-Martinez y cols, 2007). El ajo (Allium sativum) y la cebolla (Allium cepa) son dos ingredientes que han sido usados ampliamente en la gastronomía, pero también han sido utilizados como plantas medicinales durante 4000 años contra cefaleas, mordeduras de reptiles, tumores intestinales (Block, 1985). Durante los primeros juegos olímpicos en Grecia, los atletas ingerían ajo como estimulante; en la India el ajo fue usado durante siglos como loción antiséptica para el lavado de heridas y úlceras; en China el té de ajo y la cebolla han sido recomendados ampliamente contra la fiebre, el cólera y la disentería; así como en la segunda guerra mundial el ajo fue utilizado como antiséptico en prevención de la gangrena (Corzo-Martínez y cols., 2007). Además, se han estudiado los extractos de ajo y cebolla por ser efectivos en enfermedades cardiovasculares, por sus efectos hipocolesterolemiante, hipolipidémico, antihipertensivo, antidiabético y antitrombótico (Corzo-Martinez y cols, 2007), así como por su actividad en la disminución de la glucosa en sangre en ratas (Eidi y cols., 2006; Bokaeian y cols., 2010). También se ha utilizado como tratamiento en asmáticos y enfermos con dolencias broncopulmonares (Lanzotti, 2006). Introducción/Introduction 39 2.3 Compuestos organosulfurados presentes en aliáceas con potencial uso en la industria alimentaria Existen suficientes evidencias científicas que atribuyen las funciones biológicas del ajo y de la cebolla a su alto contenido en componentes organosulfurados (OS) (Augusti y Mathew, 1974). El constituyente primario en ambos vegetales es el sulfóxido derivado de la S-alquil-L-cisteína (ACSOs), precursor de los componentes lacrimógenos y el olor peculiar del género Allium (Rose y cols., 2005), como la metiína (S-metil-L- cisteinilsulfóxido y la alliína (S-alil-L-cisteinilsulfóxido), éste último es el componente mayoritario de ambos vegetales (Corzo-Martínez y cols., 2007). Hasta la fecha, cuatro componentes mayoritarios y dos minoritarios procedentes del cisteinilsulfóxido han sido identificados en el género Allium, y a partir de estos dos pueden ser generados aproximadamente 50 compuestos OS (Rose y cols,. 2005), diferentes enzimas, 17 aminoácidos y minerales como el selenio (Bhandari, 2012). La gamma-glutamilcisteína, además de servir como importante péptido de almacenamiento, es un compuesto intermedio biosintético para ACSOs (Figura 3). A partir de éste y por diferentes vías metabólicas en cada vegetal, se originan componentes volátiles. Introducción/Introduction 40 Figura 3. Formación de OS en la ruta de producción del ajo. (Corzo-Martínez, 2007) En el caso del ajo, cuando éste es cortado, machacado, masticado o deshidratado las enzimas citosólicas como la alinasa, rompen los grupos cisteínsulfóxidos, dando lugar a componentes minoritarios como los ácidos sulfénicos e intermediarios reactivos organosulfurados, siendo el ácido sulfénico el precursor intermediario de los tiosulfinatos (Musah y cols., 2016). El ácido 2-propenelsulfénico, por autocondensación da lugar a tiosulfinatos, como la alicina, dialilsulfuro (DAS), dialildisulfuro (DADS), dialil trisulfuro (DATS), ditienos, ajoenos, etc. (Lancaster y Shaw, 1989). Estos compuestos proporcionan al ajo y la cebolla su olor y sabor característicos, así como la mayor parte de sus propiedades biológicas (Lanzotti, 2006). Uno de los principales problemas de los componentes OS es su volatilidad, la alicina es conocida por ser un intermediario en la rápida descomposición en otros derivados OS, lo que indica que algunos de ellos no se encuentran originariamente en estos vegetales sino que se forman posteriormente (Corzo-Martínez y cols., 2007). Introducción/Introduction 41 En el caso de la cebolla, se parte del mismo promotor, gamma-glutamilcisteína, que por oxidación da lugar al ACSOs, y a tres sulfóxidos presentes en el tejido vegetal que fueron identificados en el laboratorio de (Virtanen y Matikkala, 1959) como son el (+)-S- S- metil-L-cisteinilsulfóxido (metiína;MCSO), (+)-S-propil-L-cisteinilsulfóxido (propiína;PCSO) y (+)-S-trans-1-propenil-L-cisteín sulfóxido o isoaliína (TPCSO) (Figura 4). La isoaliína es el mayor sulfóxido presente en la cebolla intacta y es el responsable del poder lacrimógeno de la misma (Rose y cols., 2005). Estos componentes son inestables y son rápidamente degradados transformándose en otros compuestos, en este caso también tiosulfinatos procedentes del ácido sulfénico que darán lugar a otros componentes minoritarios, bisulfuros que se oxidarán a tiosulfonatos, mono y disulfuros, como el DPS, DPDS, tiofenos, di y trisulfuros, alil metil trisulfuro (AMTS), dialil tetrasulfuro (DATeS), alil metil tetrasulfuro, dimetil trisulfuro (DMTS), dialil sulfuro (DAS) (Corzo-Martínez y cols., 2007). Introducción/Introduction 42 Figura 4. Formación de componentes organosulfurados en rutas metabólicas en cebolla procesada (Corzo- Martínez, 2007) Atendiendo a las propiedades de estos OS minoritarios y a los estudios realizados anteriormente, en el caso del ajo, numerosos OS como el DADS, DAS, DATS y el alilmetil trisulfuro y dialil tetrasulfuro han sido estudiados por su actividad antiviral y antioxidante (Romeilah y cols., 2010; Tsai y cols., 2013). Algunos autores como Llana-Ruiz-Cabello y cols., 2015a) llevaron a cabo por primera vez la evaluación de dos OS, concretamente DPDS y DPS, presentes en el aceite esencial del ajo y de la cebolla, en células de adenocarcinoma de colon Caco-2. Por otro lado, como se ha mencionado anteriormente el ajo y la cebolla han sido utilizados ampliamente en la cocina tradicional. Es por ello que estos componentes han sido estudiados por su capacidad antimicrobiana, con vistas a ser utilizados en la industria Introducción/Introduction 43 alimentaria dentro de los envases activos (Appendini y Hotchkiss, 2002). Sin embargo, y teniendo en cuenta todas las propiedades beneficiosas atribuidas al ajo, un consumo excesivo puede causar problemas tales como lesiones del tracto gastrointestinal, reacciones alérgicas, anemia, pérdida de peso, efectos tóxicos para el corazón, el hígado y los riñones, así como roturas en los cromosomas (Amagase y cols., 2001). Además, se sabe que un consumo excesivo de ajo puede causar reacciones adversas. Es por ello que se recomienda una ingestión diaria máxima de uno o dos dientes de ajo al día en adultos (Bhandari, 2012). 2.4. Propil propano tiosulfinato (PTS), propil propano tiosulfonato (PTSO). La pureza y la composición de los AEs y extractos de la especie Allium están directamente relacionadas con el ciclo vegetativo, la composición del suelo, el periodo de recogida o el tiempo de almacenamiento (Bakkali y cols., 2008), unido todo ello a la volatilidad de estos compuestos anteriormente descrita. Además, como se ha comentado previamente, estas sustancias son muy inestables y bajo diferentes condiciones los tiosulfinatos pueden descomponerse para formar OS adicionales (Benkeblia y Lanzotti, 2007). Esta variabilidad en el extracto podría afectar a sus propiedades biológicas. Con objeto de evitar estos problemas, el propil propano tiosulfinato (PTS), y el propil propano tiosulfonato (PTSO) (Figura 5) son dos OS obtenidos por descomposición del ácido sulfénico, el cual ha sido estabilizado y caracterizado por la empresa DOMCA (DMC, Granada, España). a) b) Figura 5. Estructuras químicas: a) Propil propano tiosulfonato, b) Propil propano tiosulfinato Introducción/Introduction 44 Se conoce poco sobre el PTS, pero ha mostrado efectos beneficiosos como inhibidor de la metanogénesis en rumiantes (Martínez-Fernández y cols., 2013, 2015). El PTS es considerado como un metabolito secundario del ajo, el cual actualmente se ha estudiado para evaluar su poder antimicrobiano frente la microbiota fecal en cerdos (Ruiz et al., 2015), o como tratamiento dosis-dependiente contra esporozoitos invasivos y estimulando la proliferación de células del bazo en pollos (Kim et al., 2013). Sin embargo, no se conocen sus efectos ni su seguridad sobre la salud humana. Por otro lado, el propil propano tiosulfonato (PTSO), aislado y estabilizado igualmente por DOMCA (DMC, Granada, España) ha sido previamente estudiado ya que ha inducido cambios en la composición de la microbiota intestinal, menor recuento de patógenos, y la mejora de los parámetros productivos/fisiológicos en pollos de engorde in vivo (Orban y cols., 1997; Peinado y cols., 2012, 2013); además, las dietas que contienen PTSO también dieron lugar a un aumento de la superficie de absorción a nivel ileal (Peinado y cols., 2012). Estudios llevados a cabo por nuestro equipo de investigación han demostrado que el PTSO fue capaz de inhibir el crecimiento de bacterias Gram-negativas y Gram-positivas así como hongos y levaduras (Llana-Ruiz-Cabello y cols., 2015b) Por su parte, retomando el papel de los compuestos antimicrobianos/antioxidantes utilizados en los envases activos, derivados de especies de Allium, están comercializados para su uso en la industria alimentaria, también como saborizantes o potenciadores del sabor, como ajo en polvo, cebolla cortada congelada, etc. La inclusión de un nuevo componente destinado al contacto con alimentos, además de deber cumplir unas leyes tanto europeas como españolas, deben ir acompañados de estudios toxicológicos exhaustivos que garanticen su seguridad para los consumidores. Introducción/Introduction 51 cols., 2000). Es por ello que este ensayo esté considerado como un test de elección recurrente para investigar el daño y la reparación del ADN en una amplia variedad de células tumorales o no tumorales con una amplia variedad de agentes que dañan el ADN (Liao y cols., 2009). En el caso de obtener resultados contradictorios o positivos de algún ensayo realizado in vitro, se debe proceder al estudio de genotoxicidad in vivo de la sustancia a través del ensayo de MN (OECD, 474), el ensayo cometa (OECD, 489), o el ensayo de mutación en genes transgénicos (OECD, 488). El objetivo principal del ensayo de genotoxicidad in vivo es investigar, confirmar, identificar y eliminar las sustancias que hayan podido dar falsos positivos in vitro. El ensayo in vivo de MN en eritrocitos de sangre periférica o médula ósea en mamíferos cubre los objetivos en la evaluación de CA estructurales y numéricas (daño cromosómico) y es un ensayo apropiado para la detección de componentes clastogénicos y aneugénicos. Por su parte, el ensayo cometa es un buen complemento para el estudio de la genotoxicidad, estudia el daño en el ADN, especialmente en los órganos diana que no pueden ser estudiados por otros métodos. El ensayo de roedores transgénicos puede detectar mutaciones y pequeñas delecciones de material genético. Por último, se recomienda el ensayo de síntesis de ADN no programada (UDS) en las células del hígado de mamíferos in vivo (OCDE TG 486). El hecho de combinar ambos ensayos in vitro e in vivo, se realiza de acuerdo con la guía de genotoxicidad de la Conferencia Internacional en Armonización (ICH) S2 y de la EFSA aplicando el principio de las 3 Rs: Reemplazo, Reducción y Refinamiento (EFSA, 2011). Se pretende reemplazar los animales de experimentación utilizando métodos alternativos en este caso líneas celulares. Se complementa con el estudio de varias dianas tanto en tejidos como a nivel celular in vitro, siendo especialmente útil la detección del órgano diana y el comportamiento del tóxico de estudio en los órganos asemejando las condiciones fisiológicas. El ICH recomienda una evaluación in vivo de dos tejidos diferentes, normalmente un ensayo de MN usando células hematopoyéticas de ratón y generalmente un segundo ensayo que detecte roturas en las hebras de ADN en otros tejidos mediante el empleo del ensayo cometa. Introducción/Introduction 52 Atendiendo a los resultados obtenidos en los ensayos in vitro/in vivo, se procederá a la realización de una estrategia determinada para la evaluación de la sustancia (Figura 6). Por lo tanto, si los dos ensayos in vitro son negativos, se considerará que la sustancia no tiene potencial genotóxico. Si la sustancia de estudio presenta al menos uno de los dos ensayos positivos, se procederá al estudio de los ensayos in vivo anteriormente mencionados. Tras la realización de estos ensayos, si el resultado es negativo, la sustancia será considerada sin potencial genotóxico. Si los resultados son positivos, la sustancia se considerará con potencial genotóxico. Por último, en el caso de resultados inconclusivos o contradictorios, se llevarán a cabo más estudios in vitro con el fin de esclarecer el potencial genotóxico o no de la sustancia. Por otro lado, tras la realización de los estudios in vivo, en el caso de resultados inconclusivos, contradictorios o inductores a equívoco se recomienda llevar a cabo otros estudios in vivo de genotoxicidad. Si el ensayo es negativo, puede ser considerado que la sustancia no es genotóxica; sin embargo, en algunos casos, un segundo ensayo in vivo se podría ser realizar en otro tejido para confirmar este resultado. (EFSA, 2011). Figura 6: Representación de la estrategia recomendada para la evaluación de la genotoxicidad de una sustancia. Tomada de EFSA (2011). Introducción/Introduction 53 Como se ha comentado y siguiendo las recomendaciones de las Autoridades Europeas, el ensayo de MN en la médula ósea es un buen complemento del ensayo cometa. Esto es debido a que por un lado, el ensayo de MN detecta sustancias que causan daños citogenéticos que dan lugar a la formación de MN. Estos MN proceden de fragmentos o de cromosomas enteros, fragmentados presumiblemente por la sustancia de estudio. Por su parte, el ensayo cometa in vivo es un ensayo simple en el que se puede conocer el daño en el ADN a nivel de cada célula aislada. Este ensayo en condiciones alcalinas permite detectar un rango de sustancias con potencial genotóxico incluyendo aquellas capaces de provocar roturas simples y dobles en las hebras de ADN (Bowen y cols., 2011). En segundo lugar, si consideramos que la sustancia de estudio está en el nivel 2, la EFSA recomienda además un estudio de toxicidad oral de 90 días en roedores (OECD 408). Los nuevos parámetros engloban otros anteriores presentes en el estudio de dosis repetidas de 28 días (OECD 407), pero incluyen también la determinación de hormonas tiroideas, necropsias, histopatología de tejidos que son indicadores de relaciones dosisefecto endocrinas. Los estudios de toxicidad subcrónica generalmente permiten aportar suficiente información para establecer un perfil toxicológico de la sustancia de estudio, aportando información por ejemplo, de los órganos diana y los tejidos afectados, de la naturaleza y la severidad de los efectos inducidos y de las relaciones dosis-respuesta (EFSA, 2016). Los estudios que se llevarían a cabo según esta dosis de exposición serían: (1) Estudio de toxicocinética ADME (OECD 417), da lugar a datos de absorción, distribución, metabolismo y excreción de la sustancia con la consideración de que la acumulación en el organismo podría ser anticipada. Si existen datos de actividad endocrina que sugiera un potencial efecto de la exposición prenatal, debería considerarse un estudio con un tratamiento prenatal(2) o estudio de toxicidad en la reproducción de una generación (3). Introducción/Introduction 54 (2) Estudio prenatal (OECD 414) en ratas o conejos: identifica la toxicidad potencial de una sustancia que causan efectos letales, teratogénicos y otros efectos en embriones y fetos, examinando restos fetales, embriones o muertos, así como el peso de los fetos, ratio de sexos y morfología esquelética y visceral interna y externa. Además, se han incorporado protocolos para aumentar la información toxicológica de corta duración (OECD 407) y toxicidad reproductiva con efectos potenciales en el sistema endocrino, nervioso y reproductivo. (3) Estudios de toxicidad reproductiva (OECD 443) o “extended one-generation reproduction toxicity study” (EOGRTS): aporta información sobre los efectos en la líbido y la fertilidad del macho y la hembra; en la hembra se determina la habilidad para llevar a término los embarazos, lactancia y cuidado de las crías, su tasa de supervivencia prenatal, postnatal y en el crecimiento, del desarrollo del comportamiento y la capacidad de reproducción de la descendencia, así como su mayores órganos diana (incluidos los órganos reproductores) de los progenitores y de la descendencia. Este estudio debe identificar las sustancias químicas con potencial para causar neurotoxicidad, efectos en órganos inmunológicos o reproductivos. En caso de potenciales efectos prenatales, identificados o sospechosos, se debería considerar además una exposición prenatal. También existen los estudios multigeneracionales (OECD 416). Otros estudios, correspondientes al nivel 3 son los siguientes:  Estudios de toxicidad crónica (12 meses) (OECD 452) y carcinogenicidad en roedores (OECD 451), o un estudio combinado (OECD 453). Al igual que para el estudio de la toxicidad subcrónica, en el caso de que se identifiquen o se sospechen efectos potencialmente en prenatales (por ejemplo endocrino), se recomienda una exposición prenatal. Por último, si existen datos que sugieran un potencial riesgo de neurotoxicidad o inmunotoxicidad, se deberían realizar estudios adicionales. En ese caso se recomiendan los siguientes ensayos: Introducción/Introduction 55  Estudios de potencial neurotóxico, de acuerdo con el protocolo (OECD 424)  Estudios de efectos en el desarrollo del comportamiento y la neurotoxicidad (OECD 426)  Estudios para caracterizar efectos inmunotóxicos e inmunomodulatorios, son estudios específicos de acuerdo con la guía WHO de evaluación de riesgos químicos (WHO, 2012). 3.2 Estudios de citotoxicidad y muerte celular in vitro de OS Teniendo en cuenta que entre los datos toxicológicos requeridos en el nivel 1 que propone la EFSA, se encuentra la búsqueda bibliográfica de información disponible sobre los efectos tóxicos del compuesto a considerar, se presentan a continuación los datos toxicológicos más relevantes de los componentes sulfurados que se han publicado hasta el momento. Comenzamos con los estudios de citotoxicidad que son ensayos básicos en los estudios de toxicidad in vitro. La citotoxicidad es considerada como la habilidad intrínseca de un componente para causar la muerte celular como consecuencia del daño a funciones celulares básicas (Maisanaba y cols., 2016). Si un compuesto tóxico es administrado de forma aguda in vitro, su comportamiento será anticipado en la mayoría de los casos, reflejando lo que ocurrirá en las funciones intrínsecas de las células (Eisenbrand y cols., 2002), prediciendo sus efectos agudos en organismos in vivo. Por esta razón, los ensayos de citotoxicidad son muy útiles para definir la toxicidad basal, además de ser necesarios para el establecimiento de la concentración efectiva media (CE50), concentración a la cual el 50% de las células están afectadas (Eisenbrand y cols., 2002). Esta CE50 permite definir el rango de concentraciones para la realización de otros ensayos de getoxicidad/mutagenicidad y garantizar la seguridad de estas sustancias antes de ser incorporadas en los envases activos (Maisanaba y cols., 2016). Son diversos los métodos que podemos utilizar para evaluar el descenso de la viabilidad, por ejemplo: los cambios en la permeabilidad celular son medidos por la exclusión del colorante azul tripan (Strober, 2001); el ensayo de rojo neutro (RN) que Introducción/Introduction 56 constata el daño lisosomal en la captación del colorante (Borenfreud y Puerner, 1984); la reducción de bromuro de 3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol (MTT) y sal de tetrazolio MTS (MTS) (Baltrop y cols., 1991) que determina la funcionalidad mitocondrial tras la exposición al compuesto; liberación de la enzima lactato deshidrogenasa (LDH) para evaluar el daño en la membrana celular y la valoración del contenido proteico (CP) (Bradford, 1976). Dentro de los OS, Llana-Ruiz-Cabello y cols. (2015a) estudiaron la citotoxicidad de los componentes DPDS, DPS y la mezcla de ambos en células Caco-2, utilizando biomarcadores como RN, CP y la reducción de sales de MTS alcanzando una concentración máxima de 200 μM en ambos compuestos por separado durante 24 y 48h. Las células Caco-2 expuestas al DPS no sufrieron ningún cambio respecto al control negativo tras la exposición a 24 y a 48h, en ninguno de los biomarcadores ensayados. En las células expuestas con DPDS sólo se observaron cambios en el CP que mostró un aumento significativo a la concentración más elevada utilizada en los dos tiempos de exposición. Además tampoco se observaron cambios significativos en ningún marcador en la mezcla 1:1 de los dos compuestos. Sin embargo, el PC mostró un aumento significativo a la concentración más alta (200 μM) en DPDS y en la mezcla, ambos tras 24 h de exposición. La citotoxicidad del PTSO se evaluó mediante los marcadores RN, MTS y CP en células Caco-2 y Hep-G2 a diferentes concentraciones durante 24 y a 48h, el cual indujo daño celular a partir de 350 µM en Caco-2 y Hep-G2 siendo el biomarcador más sensible el RN (EC50: 368 µM) en las células Caco-2 (Llana-Ruiz-Cabello y cols., 2015b). Por otro lado, de Martino y cols. (2006) estudiaron la toxicidad de DADS, y de dos extractos de ajo deshidratado: un extracto de ajo (GE) Latina (GEL) o Sulmona (GES) dependiendo de la región de Italia de recolección. En este estudio, el DADS fue expuesto a una concentración de 50 μM, y GES y GEL fueron expuestos al 0,1%, 0,5%, y 1%. Utilizando el azul tripán (0,2%) se determinó la viabilidad de las células a 24 y a 48h. En el caso del DADS, a 24h se produjo una disminución de la viabilidad que fue recobrada a las 48h. Los datos más relevantes respecto al GES y al GEL se observaron al 1% con una disminución de la viabilidad celular tras 24 h de exposición de ambos compuestos. Estos datos están Introducción/Introduction 57 de acuerdo con los demás ensayos realizados sobre estas células, donde se expusieron a anexina V-FITC que reacciona con la fosfatidilserina de las células apoptóticas, detectándose por citometría de flujo. Se mostró un incremento de la apoptosis en ambos compuestos y un menor valor en el caso del DADS, así como un alargamiento de la duración de las zonas de control del ciclo celular (G1), una disminución de la viabilidad y un aumento del número de células apoptóticas tanto en GES como en GEL. Por otro lado, Xiao y cols. (2004), estudiaron la inducción de la apoptosis mediante mediadores de la deoxinucleotidil transferasa mediada por dUTP, técnica (TUNEL) y por microscopía de fluorescencia tiñendo las muestras con (4',6-diamidino-2- fenilindol) (DAPI) de OS como el DATS tras 24 h de exposición, con una EC50 de 22 μM, junto con el DAS y el DADS. En este estudio se demostró que el DATS es un mayor inductor de la apoptosis en las células PC-3 asociados a la fosforilación de Bcl-2, e interaccionando con Bax, procaspasas-9 y -3. Por su parte, en las células de cáncer de mama humano, MDA-MB-231, MKL-F, KPL-1 y MCF-7. Se utilizó el ensayo de MTT, usando el rango de 1 a 100 μM de DADS durante 24, 48 y 72 h. El DADS indujo la apoptosis a concentraciones entre 1,8-18,1 μM durante 72h de exposición (Nakagawa y cols., 2001). De forma similar, se observó inducción de la apoptosis y un aumento de la caspasa-3 en las células SW-480 (cáncer de colon humano) tras el tratamiento de un OS análogo de la S- alil mercaptocisteína (Shirin y cols., 2001). Por último, otros autores han descrito que DAS y DADS podrían inducir, en muchos tipos celulares, apoptosis a través de la generación de especies reactivas de oxígeno (ERO) y consecuentemente estrés oxidativo (Truong y cols., 2009; Yi y Su, 2013). Teniendo en cuenta estos resultados tan diferentes, se hace necesario estudiar la toxicidad caso por caso antes de ser utilizados en la industria alimentaria, ya que cada tipo de OS presenta unos efectos tóxicos muy dispares. 3.3 Estudios de Genotoxicidad/Mutagenicidad in vitro Introducción/Introduction 58 Como se ha descrito anteriormente los ensayos de mutagenicidad/genotoxicidad más frecuentes descritos por la EFSA (EFSA, 2011), son el Test de Ames, el ensayo MN, y el ensayo cometa (Fig. ). Además la ICH también sugiere la realización de estos ensayos para aumentar la sensibilidad en la detección de carcinógenos, ya que no existe un sólo ensayo capaz de detectar todos los mecanismos genotóxicos relevantes en la tumogénesis (ICH, 2011). Debido a la variedad de mecanismos que conducen al daño del ADN inducido por xenobióticos y el abanico de acontecimientos mutagénicos que pueden ocurrir como resultado, se requiere de una batería de ensayos para el establecimiento del potencial genotóxico de una sustancia que se encuentre bajo investigación (Doak y cols., 2012). Entre los efectos que podemos encontrar por exposición a concentraciones subcitotóxicas de productos químicos se encuentra la interacción directa con el ADN, traduciéndose dicha interacción en diversos tipos de daño, incluyendo lesiones promutagénicas que provocan la activación de protooncogenes y la activación de genes supresores de tumores (Kensler y cols., 2003). En los ensayos de genotoxicidad, se reconoce cada uno de los tres niveles de mutación: genes, cromosomas y las mutaciones del genoma que pueden desempeñar un papel en trastornos hereditarios y en cáncer (Eisenbrand y cols., 2002). a) b) c) Figura 7. Visualización del resultado del a) Test de Ames, b) Micronúcleos y c) Ensayo Cometa. Existen pocos ensayos que hayan revelado la ausencia de mutagenicidad de los OS. Entre ellos, se han estudiado algunos componentes como el DPDS, DPS y la mezcla de ambos, (0-200 μM) los cuales fueron expuestos en el test de Ames, llevado a cabo en 5 cepas diferentes de S. typhymurium: TA97, TA98, TA100, TA102 y TA104. No se demostró que ninguno de estos componentes tuvieran actividad mutagénica en ninguna de las cepas tratadas, en presencia ni en ausencia de la fracción microsómica S9 (Llana-Ruiz- a Introducción/Introduction 59 Cabello y cols, 2015a). De la misma manera, el alil propil sulfuro (APS) y el DAS no exhibieron potencial mutagénico en el test de Ames, (TA98, TA100, TA1535, TA1537 and TA1538) (Eder y cols., 1982). Además, el extracto del ajo fresco no demostró mutagenicidad usando las cepas de S. typhimurium TA98, TA100 y TA1537 (Yoshida y cols., 1984). Por otro lado, los OS han demostrado una amplia actividad antimutagénica in vitro cuando se induce la mutagenicidad con otros compuestos. Es el caso de los extractos de ajo digeridos además de diferentes OS como DAS, DADS, DPS y DPDS evaluados por el test de Ames (TA98 y TA100) (Guyonnet y cols., 2000,2001). Los inductores de la mutagenicidad fueron: benzo[a]pireno (BaP), ciclofosfamida (CP), dimetilnitrosamina (DMN), N-nitrosopiperidina (N-PiP) y 2-amino-1-metil-6-fenilimidazo[4,5-b]piridina (PhIP). En este estudio, DAD, DPS y DPDS, promovieron la activación de compuestos promutagénicos mediados por S9 y microsomas. En el caso del DADS, solo aumentó la mutagenicidad de uno de los componentes (PhIP), por lo que mostró tener una actividad potenciadora de la mutagenicidad. Además, otros autores, estudiaron la capacidad antimutagénica del ajoeno en dos cepas de S. typhimurium TA 98 y TA 100, induciendo la mutagenicidad a partir del BaP y del 4-nitro-l,2-fenilenediamina (NPD) de una forma dosisdependiente. Particularmente, el ajoeno suprimió más efectivamente la mutagenicidad del NPD que del BaP (Ishikawa y cols., 1996). Por último, también se ha estudiado la antimutagenicidad de extractos de etil acetato procedentes de cebollas rojas, amarillas y blancas, utilizando dos cepas de S. typhymurium, TA 98 y TA100 frente a 2,2-diphenyl- picrylhydrazyl (DPPH) y H2O2 (Shon y cols., 2004). Estudios del DAS y el DADS en células cancerosas de Hámster chino (CHO) indicaron que estos componentes mostraron diferencias significativas en el ensayo de CA e intercambio de cromátidas hermanas (SCE). En presencia de la fracción microsómica S9, las CA aumentaron con el DDS pero no con DAS. El S9 redujo la inducción de SCE en ambos compuestos (Musk y cols., 1997). Los estudios realizados con MN in vitro utilizando OS son prácticamente inexistentes. Sin embargo, por ejemplo, otros AEs como lavanda y sus componentes principales: acetato de linalol y linalil fueron estudiados por este ensayo en linfocitos Introducción/Introduction 60 humanos (Di Sotto y cols., 2011). En el rango de concentraciones (0,5–100 μg/ml) el acetato de linalil aumentó la frecuencia de MN significativamente y en una concentración dosis dependiente. Por su parte, el aceite de lavanda aumentó la frecuencia de MN significativamente pero sólo a la más alta concentración. Por otro lado, el linalol demostró no ser genotóxico en ninguna de las concentraciones ensayadas. Hasta la fecha se han realizado pocos ensayos utilizando el ensayo cometa para evaluar la genotoxicidad de los OS. Sin embargo, otros AEs han sido estudiados empleando dicho ensayo cometa en linfocitos humanos (Sinha y cols., 2014). En este estudio fueron evaluados compuestos como la palmarosa, citronela o hierba del limón y geraniol (0-2000 μg/mL), además de la hierba luisa, el citral y el acetato de vetiver (0-800 μg/mL). Los resultados mostraron que la palmarosa y el aceite de citronela indujeron daño significativo en el ADN a 1000 μg/mL y concentraciones superiores; sin embargo el geraniol no mostró ninguna genotoxicidad. Por su parte, el aceite de hierba luisa y el citral exhibieron un potencial daño significativo en linfocitos humanos a partir de 100 μg/mL y 25 μg/mL respectivamente. Por último, el acetato de vetiver fue significativamente genotóxico a partir de 100 μg/mL, por lo que se confirmó su genotoxicidad. Tal y como se presentó en el caso de la mutagenicidad, también se han estudiado los efectos antigenotóxicos de los OS, estos ensayos son igualmente escasos. En primer lugar, Belloir y cols. (2006), demostraron la capacidad antigenotóxica de diferentes OS como la alicina (DADSO), el DAS, DADS, S-alil cisteína (SAC) y el alil mercaptano (AM), descritos anteriormente frente a genotoxinas, utilizando el ensayo cometa. Todos los componentes excepto el AM redujeron la genotoxicidad de la Aflatoxina B1 y el DADS fue el más efectivo reduciendo la genotoxicidad del BaP. Sin embargo, el AM y el SAC disminuyeron los fragmentos en el ADN inducidos previamente. Todos los OS disminuyeron la genotoxicidad de los componentes utilizados como control positivo, el H2O2 y el metil metano sulfonato (MMS). Por otro lado, otros componentes se estudiaron utilizando el ensayo cometa para determinar el efecto protector en el ADN de linfocitos humanos que fueron expuestos previamente al AE de Toddalia asiática (0-100 μg/mL) a los que posteriormente se les indujo la genotoxicidad con H2O2,. Los linfocitos pretratados Justificación y Objetivos/ Significance and Purposes 67 Teniendo en cuenta los antecedentes descritos previamente y tras haber estudiado los distintos tipos de envasado activo, se optó por diseñar un envase activo al que se le incorporaría AEs con propiedades antimicrobianas y antioxidantes. La aplicación de diferentes sustancias de origen natural incorporadas al envasado alimentario es de interés con el fin de aumentar la vida útil del alimento, pero puede suponer un riesgo potencial para el hombre y el medio ambiente. Este riesgo está íntimamente ligado a la migración de dichos materiales incorporados a los polímeros del envase, que serían ingeridos junto con el alimento por el consumidor. Por este motivo es necesario realizar una evaluación completa de los riesgos de la exposición humana asociados a estas sustancias, así como a los nuevos materiales de envasado que surgen tras su incorporación. Esta evaluación de los componentes requiere de una detallada caracterización del peligro potencial, conociendo los efectos tóxicos que se producirían tras la exposición de líneas celulares de órganos diana a los mismos, así como los resultados de la exposición directa en modelos experimentales in vivo, con el objetivo de poder extrapolar los datos obtenidos de la forma más certera posible al escenario de exposición humana real. En este sentido, en primer lugar, se realizó una exhaustiva revisión bibliográfica de los aceites esenciales y sus componentes mayoritarios con posibles usos en la industria alimentaria. De acuerdo con los datos disponibles, se verificó la disparidad de los resultados obtenidos, los cuales en ocasiones eran escasos y contradictorios. En segundo lugar, se consideró necesaria una revisión de los principales propiedades y efectos antimicrobianos y/o antioxidantes de los componentes mayoritarios de los AEs, así como sus propiedades mutagénicas y antimutagénicas además de genotóxicas y antigenotóxicas, y más concretamente en los componentes organosulfurados centrándonos en aquellos que se pretenden usar en la industria alimentaria. Se llegó a la conclusión de que existían determinados compuestos organosulfurados (OSCs) del género Allium, que presentaban propiedades interesantes para usos alimentarios pero que no estaban suficientemente estudiados ni evaluados para este fin. Por todo ello, nos resultó interesante evaluar la mutagenicidad/genotoxicidad del propil propano tiosulfonato (PTSO) y el propil propano tiosulfinato (PTS) mediante dos de los ensayos Justificación y Objetivos/ Significance and Purposes 68 exigidos por la EFSA: el test de Ames y el ensayo de micronúcleos (MN). Además, se han utilizado los ensayos de linfoma de ratón (MLA) y el ensayo cometa, tanto el estándar como el modificado enzimáticamente. Teniendo en cuenta que la vía de exposición humana más probable es la oral, resultó de gran interés evaluar la respuesta de líneas celulares de órganos involucrados en el proceso digestivo, destacando las células del intestino (Caco-2). Para los ensayos de genotoxicidad y mutagenicidad, las células de elección en ensayos con protocolos europeos son L5178Y TK+/-, son células de linfoma de ratón y células del sistema inmunológico humano (THP-1). Por otro lado, sería interesante detectar el posible daño genotóxico in vivo en el primer órgano de contacto, el estómago, así como el principal órgano implicado en la biotransformación de xenobióticos, el hígado, tras una exposición aguda, a las 0, 24 y 45h. Para completar estos estudios se consideró interesante determinar la posible distribución a través de la circulación sanguínea de las sustancias de estudio así como la detección de las mismas en plasma y tejidos. Una vez realizados los estudios sobre el potencial genotóxico in vitro e in vivo, se consideró interesante además llevar a cabo un estudio in vivo de toxicidad subcrónica a dosis repetidas (90 días) en ratas, basándonos en el peor escenario posible de migración al cual podría estar expuesto el consumidor. Para ello se empleó el producto comercial basado en OSCs, los cuales son característicos de las plantas del género Allium, e incorporados en un soporte inerte (ciclodextrinas) llamado PROALLIUM AP®, cuyo componente principal es el PTSO. Se realizaron estudios histopatológicos en una amplia diversidad de órganos que podrían verse afectados y se evaluaron distintos parámetros bioquímicos, hematológicos e histológicos. Por último, para la realización de esta tesis doctoral, la doctoranda realizó una estancia internacional en la que se abordó un objetivo principalmente: el aprendizaje de técnicas instrumentales como la citometría de flujo en la que se estudió el efecto en la muerte celular, estrés oxidativo y fagocitosis del PTSO en células del sistema inmunológico humano, así como la afectación del ciclo celular en las mismas tras la exposición de este compuesto. Justificación y Objetivos/ Significance and Purposes 69 Por todo ello, los objetivos específicos de la presente Tesis Doctoral han sido: 1. Realizar una exhaustiva valoración y revisión bibliográfica del estado del arte actual de los AEs destinados a su uso en la industria alimentaria. 2. Evaluar el potencial mutagénico in vitro del PTSO, en bacterias mediante el ensayo de Ames (distintas cepas de Salmonella typhimurium), y la formación de colonias en el MLA en células L5178Y TK+/-. Además, evaluar su potencial genotóxico por la formación de MN en células L5178Y TK+/- o por las roturas de las hebras de ADN (mediante el ensayo cometa) y por la posible oxidación de sus bases (mediante la versión modificada enzimáticamente) en células Caco-2. 3. Evaluar el potencial genotóxico in vivo de PTSO en ratas, estudiando la capacidad de formación de MN en eritrocitos de la médula ósea y del daño oxidativo en el ADN del órgano principal de exposición (estómago) y del principal órgano de metabolización de xenobióticos (hígado), así como la detección química del PTSO en éstos órganos y en plasma de las ratas tratadas. 4. Evaluar el potencial tóxico in vivo, mediante un ensayo de toxicidad oral subcrónica de 90 días en ratas Sprague-Dawley del PROALLIUM AP®, estudiando los cambios y alteraciones bioquímicas, hematológicas e histológicas de distintos órganos. 5. Evaluar el potencial mutagénico/genotóxico in vitro de PTS, otro componente dentro del PROALLIUM AP®, determinando su capacidad mutagénica/genotóxica a través de los cuatro ensayos anteriores. 6. Evaluar la inducción de la muerte celular, estrés oxidativo y fagocitosis, utilizando citometría de flujo en células inmunológicas humanas THP-1. El trabajo experimental se ha realizado en el Área de Toxicología de la Facultad de Farmacia de la Universidad de Sevilla, haciendo uso, así mismo del Servicio de Biología y de Microscopía del Centro de Investigación, Tecnología e Innovación de la Universidad de Sevilla (CITIUS). La obtención y aislamiento de PTSO, PTS y la incorporación de ambos en el PROALLIUM AP® fueron suministrados por DOMCA S.L. El aprendizaje de la técnica Justificación y Objetivos/ Significance and Purposes 70 de citometría de flujo ha sido realizado en el Departamento de Estrés Ambiental y Biovigilancia de los medios acuáticos (SEBIO), de la Universidad de Reims (Francia), bajo la dirección del Dr. Stéphane Betoulle y el Dr. Alain Geffard, bajo la supervisión del doctorando Hakim Samai. El análisis morfológico a nivel celular así como los estudios bioquímicos, hematológicos e histopatológicos se han realizado en colaboración con el Área de Toxicología y el Departamento de Anatomía y Anatomía Patológica Comparadas, ambos de la Universidad de Córdoba, gracias a la colaboración de la Dra. Rosario Moyano y el Dr. Alfonso Blanco. Siguiendo la normativa de la Universidad de Sevilla, el resumen, la justificación y objetivos, y las conclusiones se redactan tanto en español como en inglés para optar a la “Mención Internacional en el Título de Doctor”. Justificación y Objetivos/Objetives and Justification 67 Considering the background previously exposed and after having studied the different kind of active, we decided on the design of an active packaging that would include EOs with antimicrobial and antioxidant properties. The application of different substances from natural sources incorporated into the food packaging with the intention of increasing the shelf life of foodstuff is of interest, but it can result in potential health and environmental hazards. This risk is intimately linked to the migration of said materials incorporated into the polymers of the package, which would be ingested alongside the food by the consumer. For this reason, it is necessary to carry out a complete risk assessment associated to the human exposure to these substances, as well as the new packaging material that could appear after the incorporation. The evaluation of the components requires a detailed characterization of the potential risk, knowing the toxic effects produced after the exposure of target organ cell lines to said substances, as well as results of exposure to in vivo experimental models, with the objective of extrapolating data obtained by the most accurate way possible to the real human exposure scenario. In this sense, in the first place, an exhaustive literature study about EOs and their main components with possible uses in food industry was carried out. According to available data, the disparity of obtained results was verified, which in occasions were scarce and contradictory. In second place, a revision of the principal properties and/or antimicrobial and antioxidant effects of the main components of EOs was deemed necessary, as well as mutagenic and antimutagenic properties, specifically of organosulfur components, focusing on those with the intention of being used in the food industry. We reached the conclusion that certain organosulfur components (OSCs) of the Allium genus presented interesting properties for food uses but were not studied nor evaluated enough for this purpose. For all the reasons previously discussed, we found interesting to evaluate the mutagenicity/genotoxicity of propyl propane thiosulfonate (PTSO) and propyl propane thiosulfinate (PTS) by two of the different assays required by the EFSA: the Ames Test and the micronucleus assay (MN). In addition, the mouse lymphoma assay (MLA) and the comet assay (standard and modified) were used. Considering that oral is the most probable exposure route, Justificación y Objetivos/Objetives and Justification 68 evaluating the response of cell lines of organs involved in the digestive process was deemed interesting, highlighting intestine cells (Caco-2). For genotoxicity and mutagenicity testing, the elected cells for European protocol assays were L5178Y TK+/-, which are mice lymphoma cells, and human immune system cells (THP-1). Further on, it would be interesting to detect possible in vivo genotoxic damage in the first contact organ, the stomach, as well as the main organ involved in xenobiotic biotransformation, the liver, after acute exposure at 0, 24 and 45 hours. To complete these studies, we considered interesting evaluating the possible distribution through blood flow of target substances as well as detection in plasma and tissue. Once studies about in vivo and in vitro genotoxic potential have been performed, we also considered necessary carrying out an in vivo subchronic toxicity assay at repeated doses (90 days) in rats, based in the worst possible migration scenario that the consumer could be exposed to. To achieve this, we employed the OSC based commercial product, which are characteristic of plants from Allium genus, and incorporated it into an inert substrate (cyclodextrins) called PROALLIUM AP®, whose main component is PTSO. Histopathological studies were performed in an ample diversity of organs that could be affected and different biochemical, hematological and histological parameters were evaluated. Finally, for the fulfillment of this doctoral thesis, the PhD student went through an international research stay with one main objective particularly: the learning of instrumental techniques like flow cytometry in which the effect of cell death, oxidative stress and phagocytosis of PTSO in immune system cells was studied, as well as affliction of cell cycle after exposure to this compound. Considering this background, the main objectives of the current PhD Thesis have been: 1. To perform an exhaustive validation and bibliographical revision of current EO state-of- art destined to use in food industry. Justificación y Objetivos/Objetives and Justification 69 2. To evaluate in vitro mutagenic potential of PTSO, in bacteria by the Ames test (different Salmonella typhimirium strains) and formation of colonies in MLA in L5178Y TK+/- cells. Moreover, to evaluating its genotoxic potential by formation of MN in L5178Y TK+/- cells or by of the DNA breaks (by comet assay) and by oxidation of bases (by the enzymatic version modified) in Caco-2 cells. 3. To evaluate in vivo genotoxic potential of PTSO in rats, studying the capacity of MN formation in bone marrow erythrocytes and DNA oxidative damage in the main contact organ (stomach) and main xenobiotic metabolization organ (liver), as well as chemical detection of PTSO in these organs and in treated rat plasma. 4. To evaluate in vivo toxic potential, by a 90 days subchronical oral toxicity assay in Sprague-Dawley rats of PROALLIUM AP®, studying biochemical, hematological and histological alterations in different organs. 5. To evaluate in vitro mutagenic/genotoxic potential of PTS, another OSC, verifying in vitro mutagenic/genotoxic by means of the four aforementioned assays. 6. To evaluate cell death induction, oxidative stress and phagocytosis, using flow cytometry in human immune cells THP-1. Experimental work was carried out in the Area of Toxicology of the College of Pharmacy of the University of Seville, employing as well the Biology and Microscopy Service of the Centre of Investigation, Technology and Innovation of the University of Seville (CITIUS). The collection and isolation of PTSO, PTS and incorporation of both in PROALLIUM AP® were supplied by DOMCA S.L. Morphological analysis at ultrastructural level as well as biochemical, hematological and histopathological studies were carried out in collaboration with the Area of Toxicology and the Department of Anatomy and Pathological Comparative Anatomy, both from the University of Cordoba, thanks to collaboration with Dr. Rosario Moyano and Dr. Alfonso Blanco. The learning of flow cytometry was performed by the Department of Ambiental Stress and Biovigilance of aquatic media (SEBIO), by the University of Reims (France) under direction of Dr. Stéphane Betoulle and Dr. Alain Geffard, and under supervision of PhD student Hakim Samai. Justificación y Objetivos/Objetives and Justification 70 Following University of Seville directives, the summary, justification and objectives, and conclusions will be written both in English and Spanish to apply for “Mención Internacional en el Titulo de Doctor”. IV. Resultados y discusión / Results and discussion exposed to PTSO) were treated with medium without FBS. A negative control and a positive control (100 m MH 2 O 2 )were included to monitor the assay. The comet assay was performed as previously described by Collins et al. (1997) with modifications (Corcuera et al., 2011). Briefly, the cells were resuspended in PBS at 2.5 10 5 cells/mL and this suspension was mixed with 1% low melting point agarose and placed on a microscope slide. When gels solidified, the slides were dipped into lysis solution at 4  C for at least 1 h. Then, the nuclei were denatured in a high-pH buffer (0.3 M NaOH, 1 mM EDTA, pH13) for 20 min at 4  C in a electrophoresis tank. After electrophoresis (25 V, 20 min), the slides were neutralised in PBS and washed with deionized H 2 O(>18 M U cm-1 resistivity (Milli-Q water purification system, Millipore, Spain), dried and stained (SYBR Gold, Invitrogen, Thermofisher, MA USA). Moreover, the study of the oxidative damage in DNA by the modified comet assay was carried out using FPG and Endo III enzymes following to Llana-Ruiz-Cabello et al. (2014b). Finally, DNA was visualised (Olympus BX61 fluorescence microscope) and at least 100 randomly selected nuclei per concentration were analysed using image analysis software (Comet Assay IV, Perceptive Instruments, UK). The parameter used in the comet analysis is % DNA in tail, which was automatically obtained by this software. Three replicates of the experiment were performed. 2.8. Statistical analysis Data from the Ames test are presented as the arithmetic mean percentage ±standard deviation (SD) compared to the control group (bacterial strains exposed to sterile water). Statistical analysis was performed using the analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests using GraphPad InStat software (GraphPadSoftware Inc., La Jolla, USA). The normality of the distribution and the homogeneity of variances were confirmed using Kolmogorov and Smirnov's test, and Bartletts test, respectively. The differences were considered significant at P<0.05. In the MN tests, the statistical approach included the oneway ANOVA followed by Chi-square test. Significant differences about NDI were calculated by KruskaleWallis test (nonparametric ANOVA) between the control and treated cultures. Results were considered statistically significant from the P-value <0.05. In the MLA assay the induced mutant frequency (IMF) was determined according to the formula IMF ¼MF SMF, where MF is the test culture mutant frequency and SMF is the spontaneous mutant frequency. Positive responses are determined as those that for any treatment meet or exceed the global evaluation factor (GEF, 126 for the microwell assay) and also when a positive trend test is obtained. For the comet assays, the data (% DNA in tail) are presented as the means ±SD. The total scores of the different groups were compared using the non-parametric KruskaleWallis test followed by the ManneWhitney U-test when the first test showed differences. Differences were considered significant at P<0.05. 3. Results 3.1. Ames test After the exposure of bacterial strains to PTSO, there was no increase in the number of revertants detected in any of the strains assayed up to the highest concentration tested (20 m M), either in the presence or absence of S9 (Table 1). Toxicity against these bacterial strains, measured as a decrease in background lawn and in revertant frequency, was observed for concentrations greater than 25 m M. Positive controls produced statistically significant increases (P0.01) in the number of revertant colonies, confirming the sensitivity of the test system and the activity of the S9 mix. 3.2. Micronucleus test The concentrations to be tested in the MN test were selected according to the half maximal effective concentration (EC 50 ) values Table 1 Results of Ames test conducted with PTSO for three independent experiments. Milli Q water was used as negative control (for exposure concentrations) and pure DMSO (10 m L) as solvent for positive controls. Data are given as mean ±SD revertants/plate for three replicates for each concentration in each experiment. Positive controls without S9 for TA 97A/TA98/TA102/TA104: 2-NF (0.1 m g/plate) and for TA100: NaN 3 (1 m g/plate). Positive control for all strains with S9: 2-AF (20 m g/plate).**P<0.01 significant differences from controls. Concentration ( m M) TA97A TA98 TA100 TA102 TA104 -S9 þS9 -S9 þS9 -S9 þS9 -S9 þS9 -S9 þS9 PTSO Negative controls 169 ±24 165 ±17 22 ±315±6 108 ±9 107 ±12 252 ±54 224 ±18 236 ±17 255 ±40 1 298 ±53 239 ±26 24 ±623±6 133 ±7 138 ±13 271 ±46 380 ±19 320 ±28 331 ±48 5 196 ±40 222 ±41 25 ±622±4 142 ±15 155 ±21 292 ±26 327 ±68 279 ±26 266 ±44 10 209 ±17 225 ±56 19 ±417±4 136 ±19 120 ±5 210 ±35 288 ±55 157 ±15 244 ±48 15 180 ±15 172 ±33 21 ±422±2 117 ±798±12 187 ±25 188 ±5 327 ±5 328 ±42 20 127 ±18 87 ±620±316±6 102 ±10 101 ±6 199 ±48 139 ±28 253 ±52 359 ±88 Positive controls 680 ±71** 617 ±25** >1000** 586 ±49** 362 ±6** 322 ±22** 591 ±13** 661 ±29** 638 ±28** 752 ±6** DMSO 228 ±15 210 ±46 17 ±320±4 120 ±7 131 ±7 228 ±21 236 ±30 258 ±22 212 ±15 Table 2 Percentage of binucleated cells with micronuclei (BNMN) and the nuclear division index (NDI) in cultured mouse lymphoma cells treated with PTSO in the absence or presence of the metabolic fraction S9. Values are expressed as mean ±SD.***P<0.001 significantly different from negative control. Test substance Absence of S9 Presence of S9 Treatment time (h) Concentrations BNMN (%) ±SD NDI ±SD Treatment time (h) Concentrations BNMN (%) ±SD NDI ±SD Negative control 24 e2.3 ±0.9 1.7 ±0.1 3e6e0.8 ±0.3 1.8 ±0.1 Positive control 24 Mitomycin C 0.0625 m g/mL 6.5 ±0.9*** 1.7 ±0.1 3e6 Cyclophosfamide 8 m g/mL 3.3 ±1.5*** 1.8 ±0.3 PTSO 24 5 m M 1.9 ±0.9 1.7 ±0.1 3e6 2.5 m M 0.7 ±0.5 1.8 ±0.0 24 10 m M 2.1 ±0.7 1.7 ±0.0 3e65 m M 0.6 ±0.3 1.8 ±0.4 24 20 m M 2.2 ±0.8 1.7 ±0.1 3e610 m M 1.1 ±0.1 1.8 ±0.1 24 30 m M 1.6 ±0.9 1.7 ±0.1 3e615 m M 2.2 ±0.16*** 1.6 ±0.1 24 40 m M 2.6 ±0.7 1.6 ±0.1 3e620 m M 3.8 ±1.5*** 1.5 ±0.2 P. Mellado-García et al. / Food and Chemical Toxicology xxx (2015) 1e94 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FCT8435_proof ■22 November 2015 ■4/9 Please cite this article in press as: Mellado-García, P., et al., Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.11.011 obtained from the trypan blue exclusion assay (0e40 m M) (Fig.1). In the genotoxicity assay, PTSO was evaluated in the absence and presence of the metabolic activation fraction S9 (Table 2). Neither the frequency of binucleated cells presenting at least one micronucleus (BNMN) nor the frequency of total micronuclei (data not shown), scored per 2000 binucleated cells, showed a significant increase with respect to the control values in the case of PTSO without S9 mix. Moreover, no significant NDI variations were observed in absence of the metabolic fraction in comparison to the control group (Table 2). Nevertheless, when PTSO was tested in the presence of the S9-mix a very significant increase (P0.001) of BNMN (%) with respect to the control group was observed from 15 m M, indicating that PTSO metabolites generate a MN induction. Regarding to the NDI, significant differences (P0.001) were not observed (Table 2). As expected, treatments with MMC and CP induced significant increases in the frequency of BNMN with respect to the negative controls (P<0.001). 3.3. Mouse lymphoma assay (MLA) The results obtained in the MLA after treatment of L5178YTk þ/ cells with PTSO for 4 and 24 h are shown in Tables 3 and 4, respectively. Positive controls with MMS (10 m g/mL) were run in parallel, appearing significantly increased mutant frequencies. For PTSO 4 h assays (Table 3) previous toxicity experiments were carried out using a wide range of doses (up to 500 m M), and the range of concentrations to be used for the mutagenicity assay was selected based on the percentage of RTG, being the limit of 20% RTG widely accepted as the maximum level of cytotoxicity for mutagenicity testing to avoid biologically irrelevant effects that might occur in heavily stressed cells. In this sense, the tested concentrations ranged from 2.5 to 30 m M. At the highest concentration assayed (30 m M), a decrease in the RTG was found. However, in the 4 h-treatment assays, significant differences in induced mutation frequencies were not obtained. For the 24 h assays, PTSO was tested in the range of concentrations 1.25e20 m M, according to previous RTG values (Table 4). The observed concentration-response pattern in the decreased of RTG was similar to that 4 h-assays. However, significant values for induced mutation frequencies from 2.5 m Mto20 m M were observed in both experiments carried out. It is important to note that PTSO did not induce higher number of small colonies, being the big colonies more numerous. 3.4. Standard and enzyme-modified comet assay In the standard comet assay, results showed that PTSO (0e50 m M) did not induce DNA strand breaks in Caco-2 cells at any Table 3 Results of toxicity and mutagenicity of PTSO in mouse lymphoma cells (L5178YTk þ/ ) after 4 h of treatment.***P0.001 significantly different from negative control. Q 7 Concentration ( m M) Percent plating efficiency Mutant frequency (x 10 6 ) Relative total growth MF (S/L) a IMF (MF-SMF) Experiment I 0 130 113 100 46/67 e 2.5 104 132 87 34/98 19 5 124 163 82 39/124 50 10 93 137 62 44/93 24 20 91 187 48 58/131 74 30 111 194 47 81/113 81 MMS (10 m g/mL) 81 968*** 44 691/277 855 Experiment II 0 111 133 100 41/92 e 2.5 106 142 86 26/116 9 5 93 144 86 58/86 11 10 94 145 81 35/110 12 20 96 135 78 39/96 2 30 69 249 27 82/167 116 MMS (10 m g/mL) 75 1360*** 48 598/762 1227 a Total mutant frequency divided into small/large (S/L) colony mutant frequencies. Table 4 Results of toxicity and mutagenicity of PTSO in mouse lymphoma cells (L5178YTk þ/ ) after 24 h of treatment. *P0.05,**P0.01,***P0.001 significantly different from negative control. Percent plating efficiency Mutant frequency (x 10 6 ) Relative total growth MF (S/L) a IMF (MF-SMF) Experiment I 0 134 148 100 21/127 e 1.25 78 321 67 96/225 173 2.5 115 627** 59 176/448 479 5 118 586* 63 205/381 438 10 111 425* 39 124/289 277 20 108 466* 36 177/289 338 MMS (10 m g/mL) 34 3940*** 12 1970/1970 3792 Experiment II 0 111 166 100 70/96 e 1.25 100 302 98 91/225 136 2.5 100 408 72 171/237 242 5 104 499* 71 245/254 333 10 80 642** 69 289/353 476 20 85 734** 53 360/374 568 MMS (10 m g/mL) 54 2350*** 27 956/1434 2184 a Total mutant frequency divided into small/large (S/L) colony mutant frequencies. P. Mellado-García et al. / Food and Chemical Toxicology xxx (2015) 1e95 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FCT8435_proof ■22 November 2015 ■5/9 Please cite this article in press as: Mellado-García, P., et al., Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.11.011 of the concentrations tested after 24 or 48 h of exposure (Fig. 2A). A significant (P<0.001) increase of DNA damage was observed in Caco-2 cells exposed to 100 m MH 2 O 2 (positive control), after 24 h and 48 h of exposure, checking the effectiveness of this assay. Based on the results of the enzyme digestion of the nuclei, no increases in DNA damage was observed when Endo III (Fig. 2B) or FPG (Fig. 2C) were used in the cells exposed to PTSO after 24 and 48 h. In addition, there were no differences observed in the frequency of enzyme-sensitive sites between samples from the protein-treated and buffer-treated cells (vehicle control) (data not shown). For cells exposed to H 2 O 2 there was a significant increase in DNA breakage (P<0.001) when cells were incubated with Endo III or FPG (Fig. 2B and C, respectively). All these results indicate that PTSO did not induce primary DNA damage nor oxidized DNA bases damage at the concentrations assayed (0e50 m M) after 24 and 48 h of exposure. 4. Discussion The studies concerning the mutagenicity/genotoxicity of organosulfur compounds, main components of garlic and onion EOs, are lacking (Llana-Ruiz-Cabello et al., 2015b). Therefore such studies are needed in order to elucidate the potential risks associated with their use in the food industry. In the case of PTSO, the main component of Proallium AP ® , no data was found in the literature on the mutagenic or genotoxic potential of this compound. Previous in vitro cytotoxicity studies carried out in our laboratory in two human cell lines, Caco-2 (from human colon carcinoma) and HepG2 (from human hepatocellular carcinoma) have demonstrated that PTSO induced damage at high concentrations (the half maximal effective concentration (EC50) ranged between 350 and 415 m M, depending on the cell line, the biomarker, or the time of exposure assayed), although no significant adverse effects were recorded for the concentrations used in food packaging (Llana-Ruiz-Cabello et al., 2015a). In this work, we present results of PTSO from the four in vitro tests most commonly used for assessing the mutagenicity/genotoxicity potential of any agent. According to the genotoxicity testing strategies recommended by EFSA (EFSA, 2011) the Ames test and the MLA assay permit to investigate gene mutation induction; the MN test determine chromosome aberrations (including aneuploidy), and the comet assay, using both the standard and enzyme-modified version, determine primary DNA damage, mainly DNA breaks and oxidized DNA bases. The simultaneous use of this set of assays permits a complete assessment of the genotoxic risk of the tested compound. The range of concentrations assayed in all the experiments (0e50 m M) was limited by toxicity being in agreement with the levels expected in packaging conditions, considering the quantity of PTSO that could migrate to food from the package containing Proallium AP ® . The quantity of PTSO that would be in the package containing Proallium AP ® was calculated taking into account that only 6.5% of Proallium AP ® is added to the film and that the presence of PTSO in this extract is 14.5%. Therefore, the highest concentration of PTSO in the active packaging would be of 0.05 m M of PTSO, this is 1000-fold lower the highest concentration assayed in the present work (50 m M). In addition, considering that 5.5 g of Proallium AP ® would be added to 1 kg of ready-to-eat salad, and that a high consumer would eat 132 mg/day, (http://aesan.msssi.gob.es) the concentration that would reach the consumer is 37.5 m M which is still bellow 50 m M. The negative results obtained for PTSO in the Ames test, with and without metabolic activation, correlate well with a previous work from our laboratory. This study reported the lack of mutagenicity of other organosulfur compounds, such as dipropyl sulfide (DPS) and dipropyl disulfide (DPDS) and their 1:1 mixture, in the range of concentrations assayed (0e200 m M), in absence or presence of S9 metabolic activation (Llana-Ruiz-Cabello et al., 2015d). Moreover, fresh garlic juice and garlic extract have been demonstrated to be non-mutagenic towards TA100, TA98 and TA1537 S. typhimurium strains (Yoshida et al., 1984), and Eder et al. (1982) reported that the compounds diallyl sulfide (DAS) and allyl propyl sulfide exhibit no mutagenic activity in the Ames test (TA100, TA98, TA1537, TA1535 and TA1538). Some of these strains have GC base pairs at the primary reversion site and it is known that they may not detect certain oxidising mutagens, cross-linking agents and hydrazines (OECD, 471), and to detect cross-linking mutagens it may be preferable to include TA102, Conversely, garlic extracts were classified as positive in the Ames test in the presence of S9 mix (Kada et al.,1978). Furthermore, Shon et al. (2004) reported the antimutagenicity of ethyl acetate extracts from onions (red onions) Fig. 2. DNA damage measured in Caco-2 cells after 24 and 48 h of exposure to 5, 10, 25 and 50 m M PTSO expressed as the formation of strand breaks (a) and oxidative DNA damage as End III-sensitive sites (b) and FPG-sensitive sites (c). The levels of DNA strand-breaks (SBs), oxidized pyrimidines and oxidized purines are expressed as % DNA in tail. All values are expressed as mean ±SD. ***P<0.001 significantly different from negative control. P. Mellado-García et al. / Food and Chemical Toxicology xxx (2015) 1e96 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FCT8435_proof ■22 November 2015 ■6/9 Please cite this article in press as: Mellado-García, P., et al., Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.11.011 on S. typhimurium TA98 and TA100 strains against several model mutagens; other studies reported the dual response (mutagenicity/ antimutagenicity) of some allyl (DAS, diallyl disulfide (DADS)) and propyl (DPS, DPDS) organosulfur compounds, against some carcinogens on S. typhimurium strains (TA98, TA100) (Guyonnet et al., 2000, 2001). After application of the in vitro MN test, negative results for the induction of chromosome damage were obtained for PTSO in the experiments carried out without metabolic activation. However, the increased BNMN frequency observed in the presence of exogenous metabolic activation (S9) indicated potential genotoxicity of the metabolites. Previous studies demonstrated that other organosulfur compounds, such as DAS and DADS, were able to induce low, but significant numbers of chromosome aberrations (CA) and sister chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells (Musk et al.,1997). To understand the potential role of external metabolic activation on the genotoxicity of PTSO, our results on the MN assay can be compared with those previously obtained by the Ames test. The lack of agreement between the results obtained in both assays in the presence of S9, could be explained by assuming differences in the metabolism between bacterial and mammalian cells (Kirkland et al., 2014a) or to differences in the genetic endpoints analysed. These authors proposed that different S9 concentrations are used in the Ames test and in the in vitro mammalian cell tests and, in addition, the intrinsic metabolism of the bacterial and mammalian cells may produce different metabolites. Moreover, it also must be kept in mind that the Ames and MN tests measure different molecular endpoints, so that it is possible for a chemical to be capable of inducing chromosome breakage or nondisjunction (as measured by the MN test), but not base-pair changes, evaluated by the Ames test (Kirkland et al., 2014b). Also, it has been reported that the influence of the addition of rat liver S9 fraction to the assays modifying organosulfur compounds in a nonconsistent-manner, by enhancing the generation of chromosome aberrations by DADS, reducing the induction of SCE by both compounds (DAS and DADS), and radically altering the parameters of both survival curves (Musk et al., 1997). According to the negative results obtained by these two in vitro tests for the parent compound, in absence of S9, it can be concluded with reasonably certainty that PTSO has not genotoxic potential, at least under our testing conditions, following indications published by EFSA (2011). However, equivocal results were obtained in the presence of S9, in bacterial and L5178YTk þ/ cells. In order to clarify these results, we decided to use the in vitro MLA assay (EFSA, 2011) to add information on the potential effects at the mammalian gene mutation level. It is important to highlight that the mouse lymphoma assay (MLA) has not been used so far to study the mutagenic/genotoxic potential of EOs and their main components (Llana-Ruiz-Cabello et al. (2015b). This assay is clearly capable of detecting gene mutation, chromosomal damage and recombination induced by a wide range of chemicals. The genetic damage can be visualized by chromosome aberration analysis and also detects damage in viable cells capable of forming colonies, which is considered one of its advantages (Clements, 2000). The negative results after 4 h PTSO treatment in this mammalian cell line are in agreement with the data observed in the bacterial assays. For accomplish with the International Conference on Harmonisation (ICH) recommendations, a continuous treatment without metabolic activation for approximately 24 h is needed when negative results are obtained for the short treatment without metabolic activation. This is based upon the enhanced detection of a number of nucleoside analogues and base analogues when a 24-h treatment is included (Clements, 2000). After 24 h treatment PTSO induced significant increase in the mutation frequency indicating that prolonged exposure is needed for mutations to occur. This positive response in comparison to negative results obtained after short time exposure (4-h treatment), confirm the convincement of using 24-h treatments to detect cell-cycle specific clastogens or to enhance detection of an effect when solubility is poor or toxicity limits have been not achieved in short exposures (Clements, 2000). Finally, in order to complete the assessment of the potential genotoxicity of PTSO, the standard comet assay was performed on Caco-2 cells. While the ‘official’role of the comet assay is still limited, this test is widely used in the initial screening of pharmaceuticals, cosmetics and other chemicals for potentially mutagenic effects (Azqueta and Collins, 2013). In our work, PTSO did not induce DNA strand breaks at any concentration assayed (0e50 m M). This negative response may be because the DNA damage did not occur in vitro, or occurred and the strand breaks were easily repaired (Llana-Ruiz-Cabello et al., 2014b). The alkaline version of the comet assay (Collins et al., 1997; Singh et al., 1988) has been previously applied for EOs (Liju et al., 2013; Sinha et al., 2014; Thirugnanasampandan et al., 2012; Zegura et al., 2011) and for their main compounds used in active food packaging (Aydin et al., 2014; Martins et al., 2011; Nikolic et al., 2011; Ruiz-Cabello et al., 2014b; Saverini et al., 2012; Slamenova et al., 2013). However, for organosulfur compounds only one study has been previously carried out reporting that allicin (DADSO), DAS, DADS, S-allyl cysteine (SAC) and allyl mercaptan (AAM) did not affect cell viability of HepG2 cells nor induced DNA damage detected by the comet assay (Belloir et al., 2006). Moreover, in the present study, PTSO did not show DNA-oxidative effects when Endo III and FGP enzymes were used. This modified comet assay, if implemented in genotoxicity testing regimes, would significantly reduce the possibility of falsely classifying genotoxic compounds as negative (Azqueta and Collins, 2013). Organosulfur compounds have ben considered as antioxidant due to their reactive oxygen species scavengers properties (Llana-Ruiz-Cabello et al., 2015d). Nevertheless, it is known that antioxidant compounds could have also pro-oxidant activities, depending on the doses and conditions. From our results we can conclude that the tested compound did not act as a genotoxic agent inducing DNA breaks or oxidative DNA damage. In summary, our results show for the first time that PTSO is not mutagenic in the Ames test in presence or absence of exogenous metabolic activation (S9). Moreover the parent compound did not induce MN on mammalian cells (L5178YTk þ/ cells), while after S9 activation from 15 m M concentration PTSO generated metabolites induced chromosomal damage. Moreover, in these mammalian cells PTSO was mutagenic in the MLA test after 24-h exposure (2.5e20 m M) while after short time treatment the compound did not induce any damage. Negative results were also found in the standard comet assay in Caco-2 cells, and we also demonstrated that PTSO did not induce DNA-oxidative damage (by using the Endo-III and FPG-modified comet assay) in the range of the assayed concentrations (0e50 m M). These contradictory results for PTSO obtained from this in vitro battery assays performed, including some positive results, indicate that it may be appropriate to conduct further suitable in vivo genotoxicity tests selected on a case-by case basis, which should confirm and relate to the genotoxic endpoints identified as positive in vitro and to appropriate target organs or tissue, following the recommendations of competent authorities (EFSA, 2011). 5. Conclusion In the present study, the potential in vitro mutagenicity/genotoxicity of PTSO at relevant concentrations according to their possible use in food packaging (0e50 m M) was evaluated using a battery of genotoxicity tests. The results indicated that PTSO exhibited only a weak mutagenic potential on L5178YTk þ/ cells P. Mellado-García et al. / Food and Chemical Toxicology xxx (2015) 1e97 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FCT8435_proof ■22 November 2015 ■7/9 Please cite this article in press as: Mellado-García, P., et al., Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.11.011 after 24 h of treatment using the MLA assay, and an increased BNMN frequency was observed in the presence of metabolic activation (S9), indicating potential genotoxicity of its metabolites. Moreover, the comet assay showed that PTSO did not as a genotoxic agent inducing DNA stand breaks or oxidative DNA damage. Further in vivo genotoxicity tests are needed, such as a mammalian erythrocyte MN test (OECD 474), and an in vivo Comet assay (OECD 489), to confirm its safety before its use as active additive in food packaging. Acknowledgements The authors wish to thank the Spanish Ministry of Science and Innovation (AGL2012-38357-C02-01), co-financed with FEDER funds, and the Junta de Andalucía (AGR-7252) for the financial support of this study and Maria LLana-Ruiz Cabello also thanks Junta de Andalucia for her predoctoral grant associated to AGR- 7252. We also thanks to the Biology and Microscopy Services of CITIUS (University of Sevilla) and Floriana Mandracchia (University of Camerino, Italy) for her kind help in the laboratory. Q4 Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2015.11.011. References Amagase, H., 2006. Clarifying the real bioactive constituents of garlic. J. Nutr. 136, 716Se725S. Arranz, N., Haza, A.I., Garcia, A., Moller, L., Rafter, J., Morales, P., 2007. Protective effects of organosulfur compounds towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. Food Chem. Toxicol. 45, 1662e1669. Aydin, E., Türkez, H., Keles, M.S., 2014. The effect of carvacrol on healthy neurons and N2a cancer cells: some biochemical, anticancerogenicity and genotoxicity studies. Cytotechnology 66, 149e157. Azqueta, A., Collins, A.R., 2013. The essential comet assay: a comprehensive guide to measuring DNA damage and repair. Arch. Toxicol. 87, 949e968. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils ea review. Food Chem. Toxicol. 46, 446e475. Belloir, C., Singh, V., Daurat, C., Siess, M.H., Le Bon, A.M., 2006. Protective effects of garlic sulfur compounds against DNA damage induced by direct- and indirectacting genotoxic agents in HepG2 cells. Food Chem. Toxicol. 44, 827e834. Benkeblia, N., Lanzotti, V., 2007. Allium thiosulfinates: chemistry, biological properties and their potential utilization in food preservation. Food 1, 193e201. Cao, Y., Gu, W., Zhang, J., Chu, Y., Ye, X., Hu, Y., Chen, J., 2013. Effects of chitosan, aqueous extract of ginger, onion and garlic on quality and shelf life of stewedpork during refrigerated storage. Food Chem. 141, 1655e1660. Chung, J.G., 1999. Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in human bladder tumor cells. Drug Chem. Toxicol. 22, 343e358. Clements, J., 2000. The Mouse Lymphoma assay. Mutat. Res. 455, 97e110. Collins, A.R., Mitchell, D.L., Zunino, A., de Wit, J., Busch, D., 1997. UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts. Environ. Mol. Mutagen 29, 152e160. Collins, A.R., 2004. The comet assay for DNA damage and repair principles, applications, and limitations. Mol. Biotechnol. 26, 249e261. Corcuera, L.A., Arbillaga, L., Vettorazzi, A., Azqueta, A., L opez de Cerain, A., 2011. Ochratoxin A reduces aflatoxin B1 induced DNA damage detected by the comet assay in Hep G2 cells. Food Chem. Toxicol. 49, 2883e2889. Corzo-Martínez, M., Corzo, N., Villamiel, M., 2007. Biological properties of onions and garlic. Trends Food Sci. Technol. 18, 609e625. Echegoyen, Y., Nerin, C., 2015. Performance of an active paper based on cinnamon essential oil in mushrooms quality. Food Chem. 170, 30e36. Eder, E., Neudecker, T., Lutz, D., Henschler, D., 1982. Correlation of alkylating and mutagenic activities of allyl and allylic compounds: standard alkylation test vs. kinetic investigation. Chem-Biol. Interact. 38, 303e315. Espin, J.C., Garcia-Conesa, M.T., Tomas-Barberan, F.A., 2007. Nutraceuticals: facts and fiction. Phytochemistry 68, 2986e3008. European Food Safety Authority (EFSA), 2011. Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment. EFSA J. 9 (2379), 69. www.efsa.europa.eu/efsajournal. Last access [05/10/2015]. Evandri, M.G., Battinelli, L., Daniele, C., Mastrangelo, S., Bolle, P., Mazzanti, G., 2005. The antimutagenic activity of Lavandula angustifolia (lavender) essential oil in the bacterial reverse mutation assay. Food Chem. Toxicol. 43, 1381e 1387. Fenech, M., 2000. The in vitro micronucleus technique. Mutat. Res. 455, 81e95. Garcia, A., Haza, A.I., Arranz, N., Delgado, M.E., Rafter, J., Morales, P., 2008. Organosulfur compounds alone or in combination with vitamin C protect towards N- nitrosopiperidine and N-nitrosodibutylamineinduced oxidative DNA damage in HepG2 cells. Chem. Biol. Interact. 173, 9e18. Guyonnet, D., Belloir, C., Suschetet, M., Siess, M.H., Le Bon, A.M., 2000. Liver subcellular fractions from rats treated by organosulfur compounds from Allium modulate mutagen activation. Mutat. Res. 466, 17e26. Guyonnet, D., Belloir, C., Suschetet, M., Siess, M.H., Le Bon, A.M., 2001. Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction. Mutat. Res. 495, 135e145. Honma, M., Hayashi, M., Shimada, H., Tanaka, N., Wakuri, S., Awogi, T., Yamamoto, K.I., Kodani, N., Nishi, Y., Nakadate, M., Sofuni, T., 1999. Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test. Mutagenesis 14, 5e22. http://aesan.msssi. gob.es (accesed 20.10.05). ICH Expert Working Group, 2008. International Conferences on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, in ICH Harmonised Tripartite Guideline Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. Draft Version 5.3. Kada, T., Sadaie, Y., Hara, M., 1978. Analysis of mutagen-antimutagen reactions in food and food additives by the rec-assay and reversion-assay procederes. Mutat. Res. 53, 206e207. Kirkland, D., Zeiger, E., Madia, F., Gooderham, N., Kasper, P., Lynch, A., Morita, T., Ouedraogo, G., Parra Morte, J.M., Pfuhler, S., Rogiers, V., Schulz, M., Thybaud, V., van Benthem, J., Vanparys, P., Worth, A., Corvi, R., 2014a. Can in vitro mammalian cell genotoxicity test results be used to complement positive results in the Ames test and help predict carcinogenic or in vivo genotoxic activity? I. Reports of individual data bases presented at an EURL ECVAM Workshop. Mutat. Res. Genet. Toxicol. Environ. Mutagen 775e776, 55e68. Kirkland, D., Zeiger, E., Madia, F., Corvi, R., 2014b. Can in vitro mammalian cell genotoxicity test results be used to complement positive results in the Ames test and help predict carcinogenic or in vivo genotoxic activity? II. Construction and analysis of a consolidated data base. Mutat. Res. Genet. Toxicol. Environ. Mutagen 775e776, 69e80. Le Bon, A.M., Siess, M.H., 2000. Organosulfur compounds from Allium and the chemoprevention of cancer. Drug Metab. Drug Interact. 17, 51e79. Lee, S.Y., Lee, S.J., Choib, D.S., Hura, S.J., 2015. Current topics in active and intelligent food packaging for preservation of fresh foods. J. Sci. Food Agric. http:// dx.doi.org/10.1002/jsfa.7218. Q 5 Liju, V.B., Jeena, K., Kuttan, R., 2013. Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric (Curcuma longa L.). Food Chem. Toxicol. 53, 52e61. Llana-Ruiz-Cabello, M., Guti errez-Praena, D., Pichardo, S., Moreno, F.J., Bermúdez, J.M., Aucejo, A., Came an, A.M., 2014a. Cytotoxicity and morphological effects induced by carvacrol and thymol on the human cell line Caco-2. Food Chem. Toxicol. 64, 281e290. Llana-Ruiz-Cabello, M., Maisanaba, S., Puerto, M., Prieto, A.I., Pichardo, S., Jos, A., Came an, A.M., 2014b. Evaluation of the mutagenicity and genotoxic potential of carvacrol and thymol using the Ames Salmonella test and alkaline, Endo III- and FPG-modified comet assays with the human cell line Caco-2. Food Chem. Toxicol. 72, 122e128. Llana-Ruiz-Cabello, M., Guti errez-Praena, D., Puerto, M., Pichardo, S., Moreno, F.J., Ba~ nos, A., Nu~ nez, C., Guillam on, E., Came an, A.M., 2015a. Acute toxicological studies of the main organosulfur compound derived from Allium sp. intended to be used in active food packaging. Food Chem. Toxicol. 82, 1e11. Llana-Ruiz-Cabello, M., Pichardo, S., Maisanaba, S., Puerto, M., Prieto, A.I., Guti errez- Praena, D., Jos, A., Came an, A.M., 2015b. In vitro toxicological evaluation of essential oils and their main compounds used in active food packaging: a review. Food Chem. Toxicol. 81, 9e27. Llana-Ruiz-Cabello, M., Pichardo, S., Ba~ nos, A., Nu~ nez, C., Bermudez, J.M., Guillam on, E., Aucejo, S., Came an, A.M., 2015c. Characterisation and evaluation of PLA films containing an extract of Allium spp. to be used in the packaging of ready-to-eat salads. LWT-Food Sci. Technol. 64, 1354e1361. Llana-Ruiz-Cabello, M., Maisanaba, S., Guti errez-Praena, D., Prieto, A.I., Pichardo, S., Jos, A., Moreno, F.J., Camean, A.M., 2015d. Cytotoxic and mutagenic in vitro assessment of two organosulfur compounds derived from onion to be used in the food industry. Food Chem. 166, 423e431. Lohani, M., Yadav, S., Schiffmann, D., Rahman, Q., 2003. Diallylsulfide attenuates asbestos induced genotoxicity. Toxicol. Lett. 143, 45e50. Maisanaba, S., Prieto, A.I., Puerto, M., Guti errez-Praena, D., Demir, E., Marcos, R., Came an, A.M., 2015. In vitro genotoxicity testing of carvacrol and thymol using the micronucleus and mouse lymphoma assays. Mutat. Res. 784e785, 37e44. Martins, C., Doran, C., Laires, A., Rueff, J., Rodrigues, A.S., 2011. Genotoxic and apoptotic activities of the food flavourings myristicin and eugenol in AA8 and XRCC1 deficient EM9 cells. Food Chem. Toxicol. 49, 385e392. Milner, J.A., 2006. Preclinical perspectives on garlic and cancer. J. Nutr. 136, 827Se831S. Musk, S.R.R., Clapham, P., Johnson, I.T., 1997. Cytotoxicity and genotoxicity of diallyl sulfide and diallyl disulfide towards Chinese hamster ovary cells. Food Chem. Toxicol. 35, 379e385. Nerin, C., Tovar, L., Djenane, D., CamoJ, Salafranca, J., Beltr an, J.A., Rocal es, P., 2006. Stabilization of beef meat by a new active packaging containing natural antioxidants. J. Agr. Food Chem. 54, 7840e7846. P. Mellado-García et al. / Food and Chemical Toxicology xxx (2015) 1e98 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FCT8435_proof ■22 November 2015 ■8/9 Please cite this article in press as: Mellado-García, P., et al., Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.11.011 Nikolic, B., Mitic-Culafic, D., Vukovic-Gacic, B., Knezevic-Vukcevic, J., 2011. Modulation of genotoxicity and DNA repair by plant monoterpenes camphor, eucalyptol and thujone in Escherichia coli and mammalian cells. Food Chem. Toxicol. 49, 2035e2045. Nkrumah-Elie, Y.M., Reuben, J.S., Hudson, A.M., Taka, E., Badisa, R., Ardley, T., Israel, B., Sadrud-Din, S.Y., Oriaku, E.T., Darling-Reed, S.F., 2012. The attenuation of early benzo(a)pyreneinduced carcinogenic insults by diallyl disulfide (DADS) in MCF-10Acells. Nutr. Cancer 64, 1112e1121. OECD (Organisation for Economic Cooperation and Development), 1997. Guideline for Testing of Chemicals: Mutation Test, pp. 1e11. Guideline 471. OECD (Organisation for Economic Cooperation and Development), 2014. Guideline for Testing of Chemicals: in vitro Mammalian Cells Micronucleus Test, pp. 1e26. Guideline 487. OECD (Organisation for Economic Cooperation and Development), 2015. Guideline for Testing of Chemicals: In vitro Mammalian Cell Gene Mutation Test Using the Hprt and Xprt Genes, pp. 1e18. Guideline 476. Peinado, M.J., Ruiz, R., Ech avarri, A., Rubio, L.A., 2012. Garlic derivative propyl propane thiosulfonate is effective against broiler enteropathogens in vivo. Poult. Sci. 91, 2148e2157. Peinado, M.J., Ruiz, R., Ech avarri, A., Aranda-Olmedo, I., Rubio, L.A., 2013. Garlic derivative PTS-O modulates intestinal microbiota composition and improves digestibility in growing broiler chickens. Anim. Feed Sci. Technol. 181, 87e92. Ruiz, R., García, M.P., Lara, A., Rubio, L.A., 2010. Garlic derivatives (PTS and PTS-O) differently affect the ecology of swine faecal microbiota in vitro. Vet. Microbiol. 144, 110e117. Saverini, M., Catanzaro, I., Sciandrello, G., Avellone, G., Indelicato, S., Marcì, G., Palmisano, L., 2012. Genotoxicity of citrus wastewater in prokaryotic and eukaryotic cells and efficiency of heterogeneous photocatalysis by TiO 2 . J. Photochem. Photobiol. B 108, 8e15. Seow, Y.X., Yeo, C.R., Chung, H.L., Yuk, H.-G., 2014. Plant essential oils as active antimicrobial agents. Crit. Rev. Food Sci. Nutr 54, 625e644. Sheen, L.-Y., Wu, C.-C., Li, C.-K., Tsai, S.-J., 2001. Effect of diallyl sulfide and diallyl disulfide, the active principles of garlic, on the aflatoxin B1-induced DNA damage in primary rat hepatocytes. Toxicol. Lett. 122, 45e52. Shon, M.Y., Choi, S.D., Kahng, G.G., Nam, S.H., Sung, N.J., 2004. Antimutagenic, antioxidant and free radical scavenging activity of ethylacetate extracts from white, yellow and red onions. Food. Chem. Toxicol. 42, 659e666. Shukla, Y., Arora, A., Taneja, P., 2003. Antigenotoxic potential of certain dietary constituents. Teratog. Carcin. Mut. 23, 323e335. Q 6 Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184e191. Sinha, S., Jothiramajayam, M., Ghosh, M., Mukherjee, A., 2014. Evaluation of toxicity of essential oils palmarosa, citronella, lemongrass and vetiver in human lymphocytes. Food Chem. Toxicol. 68, 71e77. Slamenova, D., Kozics, K., Hunakova, L., Melusova, M., Navarova, J., Horvathova, E., 2013. Comparison of biological processes induced in HepG2 cells by tert-butyl hydroperoxide (t-BHP) and hydroperoxide (H 2 O 2 ): the influence of carvacrol. Mutat. Res. 757, 15e22. Soriano, C., Creus, A., Marcos, R., 2007. Gene-mutation induction by arsenic compounds in the mouse lymphoma assay. Mutat. Res. 634, 40e50. Strober, W., 2001. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. Appendix 3 : Appendix 3B. Thirugnanasampandan, R., Jayakumar, R., Prabhakaran, M., 2012. Analysis of chemical composition and evaluation of antigenotoxic, cytotoxic and antioxidant activities of essential oil of Toddalia asiatica (L.) Lam. Asia Pac. J. Trop. Biomed. S1276eS1279. Wang, J., Sawyer, J.R., Chen, L., Chen, T., Honma, M., Mei, N., Moore, M., 2009. The Mouse Lymphoma Assay detects recombination, deletion, and aneuploidy. Toxicol. Sci. 109, 96e105. Wilson, E.A., Demmig-Adams, B., 2007. A Antioxidant, anti-inflammatory, and antimicrobial properties of garlic and onions. Nutr. Food Sci. 37, 178e183. Yamazaki, H., Oda, Y., Funae, Y., Imaoka, S., Inui, Y., Guengerich, F.P., Shimada, T., 1992. Participation of rat liver cytochrome P450 2E1 in the activation of N- nitrosodimethylamine and N-nitrosodiethylamine to products genotoxic in an acetyltransferase-overexpressing Salmonella typhimurium strain (NM2009). Carcinogenesis 13, 979e985. Yoshida, S., Hirao, Y., Nakagawa, S., 1984. Mutagenicity and cytotoxicity tests of garlic. J. Toxicol. Sci. 9, 77e86. Zegura, B., Dobnik, D., Niderl, M.H., Filipic, M., 2011. Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells. Environ. Toxicol. Pharmacol. 32, 296e305. P. Mellado-García et al. / Food and Chemical Toxicology xxx (2015) 1e99 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 FCT8435_proof ■22 November 2015 ■9/9 Please cite this article in press as: Mellado-García, P., et al., Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.11.011 89 Capítulo II / Chapter II Pilar Mellado-García, María Puerto, Ana I. Prieto, Silvia Pichardo, Ana Martín-Cameán, Rosario Moyano, Alfonso Blanco, Ana M. Cameán GENOTOXICITY OF A THIOSULFONATE COMPOUND DERIVED FROM ALLIUM SP. INTENDED TO BE USED IN ACTIVE FOOD PACKAGING: IN VIVO COMET ASSAY AND MICRONUCLEUS TEST. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 800-801, 1-11, 2016 90 Dear Author, Please, note that changes made to the HTML content will be added to the article before publication, but are not reflected in this PDF. Note also that this file should not be used for submitting corrections. Please cite this article in press as: P. Mellado-García, et al., Genotoxicity of a thiosulfonate compound derived from Allium sp. intended to be used in active food packaging: In vivo comet assay and micronucleus test, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2016), http://dx.doi.org/10.1016/j.mrgentox.2016.03.001 ARTICLE IN PRESS G Model MUTGEN 402712 1–11 Mutation Research xxx (2016) xxx–xxx Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis jo ur nal home page: www.elsevier.com/locate/gentox Comm unit y ad dress: www.elsevier.com/locate/mutres Genotoxicity of a thiosulfonate compound derived from Allium sp. intended to be used in active food packaging: In vivo comet assay and micronucleus test Pilar Mellado-Garcíaa Q1 , María Puertoa, Ana I. Prietoa, Silvia Pichardoa, Ana Martín-Cameána, Rosario Moyanob, Alfonso Blancoc, Ana M. Cameána,∗ aArea of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n◦2, 41012 Seville, Spain bDepartment of Pharmacology, Toxicology and Legal and Forensic Medicine, Faculty of Veterinary Medicine, University of Córdoba, Campus de Rabanales, Córdoba, Spain cDepartment of Anatomy and Comparative Pathology and Anatomy, University of Córdoba, Campus de Rabanales, Córdoba, Spain a r t i c l e i n f o Article history: Received 18 November 2015 Accepted 2 March 2016 Available online xxx Keywords: Allium sp. Propyl thiosulphinate oxide Genotoxicity Comet assay Micronucleus Rat a b s t r a c t Components of Allium species have antimicrobial and antioxidant properties. A commercial Allium sp. extract (Proallium AP®), of which the main constituent is propyl thiosulphinate oxide (PTSO), is being used in the development of active food packaging. In previous in vitro genotoxicity studies, PTSO, in the presence of metabolic activation, increased the appearance of micronuclei (MN). We assessed the genotoxicity PTSO in rats following oral administration (doses: 5.5, 17.4, and 55 mg/kg). The comet assay in liver and stomach (OECD 489) and the MN assay in bone marrow (OECD 474) were carried out. After necropsy, histopathological examinations of the liver and the stomach were performed. The results revealed no in vivo genotoxicity and the histopathological analysis showed only slight modifications, such as increased glycogen storage in the liver and a degenerative process in stomach, with vacuolization of cell membranes, only at the highest dose. Therefore, the present work confirms that this compound is not genotoxic and could be considered as a natural alternative to synthetic preservatives used in the food packaging industry. © 2016 Published by Elsevier B.V. 1. Introduction Garlic (Allium sativum) and onion (Allium cepa) have been used for medical applications and as flavouring agents for hundreds of years [1]. Some of the properties of Allium species could be useful for the food industry, including their antioxidant, antibacterial, antifungal, and anti-yeast activities [2,3]. Garlic is used as a nutraceutical agent to prevent dyslipidemia, hyperglycemia, cardiovascular disorders, etc. [4]. Garlic and onion contain biologically active substances, including volatile, water-soluble, and oil-soluble organosulfur compounds (OSCs), which may be responsible for their beneficial properties [5,6]. However, the content of OSCs in Allium species can vary substantially [7]. The isolation of the compound propyl propane thiosulfonate (PTSO) affords a stable, active agent for use in the food industry. PTSO is incorporated into an inert commercial alimentary support (propylene glycol and polysorbate) ∗Corresponding author. E-mail address: [email protected] (A.M. Cameán). to produce Proallium AP®(DOMCA S.L, Granada Spain). In view of the great interest in the use of natural active compounds in the package industry and the antioxidant and antimicrobial properties of PTSO [8], Proallium AP®has been selected to be incorporated into films to produce active packaging. The purpose of such packaging is to allow the release of PTSO onto the food surface, in order to increase the shelf life of ready-to-eat salads. The effectiveness of the resulting material has been verified [9] and PTSO may be a good natural alternative to the synthetic preservatives used in the food packaging industry. Before PTSO can be commercialized for this purpose, many toxicological assays are required. Q2 Previous in vitro cytotoxicity studies carried out in our laboratory, with human intestinal and hepatic cell lines, demonstrated that although PTSO induced damage at high concentrations (350–415 ␮M), no significant adverse effects were seen at concentrations intended to be used in food packaging [8]. The European Food Safety Authority (EFSA) recommends a step-wise approach for testing the genotoxic potential of substances in food and feed, beginning with two in vitro tests: a bacterial reverse mutation (Ames test) and an in vitro micronucleus (MN) test. In the case http://dx.doi.org/10.1016/j.mrgentox.2016.03.001 1383-5718/© 2016 Published by Elsevier B.V. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Please cite this article in press as: P. Mellado-García, et al., Genotoxicity of a thiosulfonate compound derived from Allium sp. intended to be used in active food packaging: In vivo comet assay and micronucleus test, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2016), http://dx.doi.org/10.1016/j.mrgentox.2016.03.001 ARTICLE IN PRESS G Model MUTGEN 402712 1–11 8 P. Mellado-García et al. / Mutation Research xxx (2016) xxx–xxx Fig. 6. Histopathological changes in liver of rats treated with PTSO. (A, C, E) HE-stained liver section. Bars, 100 ␮m. (B, D, F) Ultrastructural observations. Bars, 10 ␮m. (A, B) Negative control rats. (A) Normal hepatic cords (He), polyhedral morphology with clear cytoplasm and nucleus. (B) Details of apparently normal hepatocytes (He) with cytoplasmic organelles, reticulum and mitochondria (circle). (C, D) Positive control rats exposed to EMS. (C) Detail of liver parenchyma with presence of atypical mitosis (Mi). (D) Detail of hepatocyte mitosis (Mi). (E, F) Rat treated with 55 mg/kg bw PTSO. (E) Hepatocytes showed no nuclear alterations, only accumulations of glycogen are observed (arrow). (F) Normal hepatocyte (He). plasma membrane in liver observed under EM, although no damage was reported at lower doses (250 and 500 mg/kg bw) [53]. The latter research group also observed focal myocytolysis in heart from rats exposed to 1000 mg/kg bw of garlic homogenate by gavage, while at lower doses they retained their normal histology [54]. In order to analyze the safety of PTSO, a comparison between previous in vitro [11] and the in vivo results obtained in this work, at their respective concentrations assayed, is necessary. The in vitro positive responses of PTSO (5–20 ␮M) evidenced by the MLA and MN assays were obtained at exposure concentrations much lower than the in vivo concentrations assayed here (highest dose of 55 mg/kg equivalent to 30 mM). These discrepancies between genotoxicity tests in mammalian cells in vitro when compared to in vivo assays have been discussed, and usually the substances were less toxic in vivo, indicating detoxification processes [49,55]. EFSA [56] indicated that over 90% of in vitro positives were “irrelevant positives” detected by mammalian cell assays under in vitro conditions. In most of the rodent cell lines used, deficiencies in metabolism, p53 function and DNA repair capability almost certainly contribute to this false positive rate; moreover, testing up to high concentrations and high levels of cytotoxicity, as is currently required in mammalian cells, was also likely to contribute to the high frequency of “false positive” results [49]. Actually, none of the factors frequently invoked to explain the high frequency of in vitro positives (high dosing, artificial metabolic conditions) seem sufficient to account for these results [55]. In our case, the discrepancy between in vitro and in vivo results obtained could be explained not in terms of concentrations, but to differences in metabolism and/or in the bioavailability of the test substance in the target organ [55]. Regarding the first aspect, the risks resulting from the formation of reactive intermediates in vitro are reduced in vivo by the presence of detoxifying systems, which are often extremely 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 Please cite this article in press as: P. Mellado-García, et al., Genotoxicity of a thiosulfonate compound derived from Allium sp. intended to be used in active food packaging: In vivo comet assay and micronucleus test, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2016), http://dx.doi.org/10.1016/j.mrgentox.2016.03.001 ARTICLE IN PRESS G Model MUTGEN 402712 1–11 P. Mellado-García et al. / Mutation Research xxx (2016) xxx–xxx 9 Fig. 7. Histopathological changes in stomach of rats treated with PTSO. (A, C, E) HE-stained liver section. Bars, 100 ␮m. (B, D, F) Ultrastructural observations. Bars, 10 ␮m. (A, B) Negative control rats. (A) Details of gastric mucosa with normal cells (M). (B) Details of the gastric mucosa with apparently normal secretion granules (circle). (C, D) Positive control rats exposed to EMS. (C) Details of the superficial mucosa with very basophils nuclei undergoing necrosis (circle). (D) Details of the principal cells with abundant phagosomes as a cellular debris by necrosis (circle). (E, F) Rat treated with 55 mg/kg bw PTSO. (E) Details of normal parietal and chief cells (circle). (B) Detail of chief cells with vacuolized membranes (circle). efficient: a) by avoiding the formation of the ultimate mutagen by metabolic sequestration of a promutagen; or b) by inactivation of the active metabolite after its formation (degradation products of PTSO detected in tissues, Fig. 5). Currently, the impact of metabolic differences between in vitro and in vivo test systems on the false positive rate in in vitro genotoxicity tests is not known [49]. With respect to bioavailability, this may be a critical factor, direct evidence of reaching the bone marrow (or blood) at biologically relevant concentrations being necessary. In this work, PTSO was not detected in blood, and this could be one reason for the apparent insensitivity of the MN assay in vivo. In tissues, the detection of two degradation products derived from PTSO in stomach of treated rats demonstrated their in vivo exposure to the compound, which seems to suffer hydrolysis reactions. In the liver of exposed rats only a single organosulfur compound has been detected in comparison to control ones, indicating the influence of distribution and metabolism processes. A study focused on toxicokinetic aspects of PTSO is needed to draw definitive conclusions. To assess substances of unknown toxicity present at low levels in the diet, the Threshold of Toxicological Concern (TTC) approach is recommended [56,57]. However, a critical aspect for an appropriate application of the TTC principle is the need for reliable exposure data of the compound [57]. In our case, this information is lacking and, consequently, the TTC approach could not yet be applied. The risk assessment of PTSO requires more compound-specific toxicity data. 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 Please cite this article in press as: P. Mellado-García, et al., Genotoxicity of a thiosulfonate compound derived from Allium sp. intended to be used in active food packaging: In vivo comet assay and micronucleus test, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2016), http://dx.doi.org/10.1016/j.mrgentox.2016.03.001 ARTICLE IN PRESS G Model MUTGEN 402712 1–11 10 P. Mellado-García et al. / Mutation Research xxx (2016) xxx–xxx 5. Conclusions Our results confirm the absence of genotoxicity of PTSO in rats, which is relevant in terms of its toxicological assessment to be used in the food packaging industry. The combined in vivo genotoxicity assays gave negative results in both the comet and MN assays. In addition, the use of enzymes which reveal oxidative damage did not indicate DNA oxidation. Finally, the histopathological study showed that the main target organs for PTSO were liver and stomach, since no damage was observed in any other tissues when rats were exposed to the highest dose assayed (55 mg/kg bw). We can conclude that PTSO is a promising active substance for use in active food packaging, but more toxicological data are required before its approval. Acknowledgements The authors wish to thank the Spanish Ministry of ScienceQ3 and Innovation (AGL2012-38357-C02-01), co-financed with FEDER funds, and the Junta de Andalucía (AGR-7252) for the financial support of this study, the Microscopy Service of CITIUS and the animal housing from the Faculty of Pharmacy in the University of Sevilla. The authors also would like to thank Dr. Remedios Guzmán (Area Toxicology, University of Sevilla) for reviewing the new version of this manuscript, to Dr. Jose Antonio González (IRNA, CSIC) for performing the pyrolytic analysis of tissues, and Adoración Puente for the technical assistance. References [1] R.M. Romeilah, S.A. Fayed, G.I. Mahmoud, Chemical compositions, antiviral and antioxidant activities of seven essential oils, J. Appl. Sci. Res. 6 (2010) 50–62. [2] J.C. Harris, S.L. Cottrell, S. Plummer, D. Lloyd, Antimicrobial properties of Allium sativum (garlic), Appl. Microbiol. Biotechnol. 57 (2001) 282–286. [3] E.A. Wilson, B. Demmig-Adams, Antioxidant anti-inflammatory, and antimicrobial properties of garlic and onions, Nutr. Food Sci. 37 (2007) 178–183. [4] H.A.R. Suleria, M.S. Butt, N. Khalid, S. Sultan, A. Raza, M. Aleem, M. Abbas, Garlic (Allium sativum): diet based therapy of 21st century—a review, Asian Pac. J. Trop. Dis. 5 (2015) 271–278. [5] H. Amagase, B.L. Petesch, H. Matsuura, S. Kasuga, Y. Itakura, Intake of garlic and its bioactive components, J. Nutr. 131 (2001) 955S–962S. [6] M. Corzo-Martínez, N. Corzo, M. Villamiel, Biological properties of onions and garlic, Trends Food Sci. Technol. 18 (2007) 609–625. [7] N. Benkeblia, V. Lanzotti, Allium thiosulfinates: chemistry, biological properties and their potential utilization in food preservation, Food 1 (2007) 193–201. [8] M. Llana-Ruiz-Cabello, D. Gutiérrez-Praena, M. Puerto, S. Pichardo, F.J. Moreno, A. Ba˜ nos, C. Nu˜ nez, E. Guillamón, A.M. Cameán, Acute toxicological studies of the main organosulfur compound derived from Allium spp. intended to be used in active food packaging, Food Chem. Toxicol. 82 (2015) 1–11. [9] M. Llana-Ruiz-Cabello, S. Pichardo, A. Ba˜ nos, C. Nu˜ nez, J.M. Bermúdez, E. Guillamón, S. Aucejo, A.M. Cameán, Characterisation and evaluation of PLA films containing an extract of Allium spp. to be used in the packaging of ready-to-eat salads under controlled atmospheres, LWT—Food Sci. Technol. 64 (2015) 1354–1361. [10] European Food Safety Authority (EFSA), Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment, EFSA J. 9 (2011) 2379–2448. [11] P. Mellado-García, S. Maisanaba, M. Puerto, M. Llana-Ruiz-Cabello, A.I. Prieto, R. Marcos, S. Pichardo, A.M. Cameán, Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging, Food Chem. Toxicol. 86 (2015) 365–373. [12] D.E. Bowen, J.H. Whitwell, L. Lillford, D. Henderson, D. Kidd, S. McGarry, G. Pearce, C. Beevers, D.J. Kirkland, Evaluation of a multi-endpoint assay in ratsQ4 combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test, Mutat. Res. 722 (2011) 7–19. [13] OECD (Organisation for Economic Cooperation and Development), Guideline for the testing of chemicals: mammalian erythrocyte micronucleus test, Guideline 474 (2014) 1–21. [14] L.A. Corcuera, A. Vettorazzi, L. Arbillag, N. Pérez, A.G. Gil, A. Azqueta, E. González-Pe˜ nas, J.A. García-Jalón, A. López de Cerain, Genotoxicity of aflatoxin B1 and ochratoxin A after simultaneous application of the in vivo micronucleus and comet assay, Food Chem. Toxicol. 76 (2015) 116–124. [15] A.R. Collins, V.L. Dobson, M. Dusinska, G. Kennedy, R. Stetina, The comet assay: what can it really tell us? Mutat. Res. 375 (1997) 183–193. [16] A.R. Collins, M. Dusinska, E. Horvathova, E. Munro, M. Savio, R. Stetina, Inter-individual differences in repair of DNA base oxidation, measured in vitro with the comet assay, Mutagenesis 16 (2001) 297–301. [17] A.R. Collins, A.A. Oscoz, G. Brunborg, I. Gaivao, L. Giovannelli, M. Kruszewski, C.C. Smith, R. Stetina, The comet assay: topical issues, Mutagenesis 23 (2008) 143–151. [18] C.W. Tsai, H.W. Chen, L.Y. Sheen, C.K. Lii, Garlic: health benefits and actions, Biomedicine 2 (2012) 17–29. [19] E.G. Yordi, E.M. Pérez, M.J. Matos, E.U. Villares, Antioxidant and pro-oxidant effects of polyphenolic compounds and structure-activity relationship evidence, in: J. Bouayed, T. Bohn (Eds.), Nutrition, Well-Being and Health, InTech, Croatia, 2012, pp. 23–48. [20] M. Carocho, C.F.R. Ferreira, A review on antioxidants, prooxidants and related controversy: natural and synthetic compounds, screening and analysis methodologies and future perspectives, Food Chem. Toxicol. 51 (2013) 15–25. [21] M. Vasquez, Combining the in vivo comet and micronucleus assays: a practical approach to genotoxicity testing and data interpretation, Mutagenesis 25 (2010) 187–199. [22] ICH (International Conference on Harmonisation). Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2 (R1). 2012. http://www.fda.gov/downloads/Drugs/Guidances/ucm074931. pdf (last access 12/11/2015). [23] OECD, (Organisation for Economic Cooperation and Development), guideline for the testing of chemicals: in vivo mammalian alkaline comet assay, Guideline 489 (2014) 1–25. [24] Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, 276 (2010) 33–79. [25] L.A. Corcuera, M. Iba˜ nez-Vea, A. Vettorazzi, E. González-pe˜ nas, A. López de Cerain, Validation of a UHPLC-FLD analytical method for the simultaneous quantification of aflatoxin B1 and ochratoxin A in rat plasma, liver and kidney, J. Chromatogr. B 879 (2011) 2733–2740. [26] P. Abad, F.J. Lara, N. Arroyo-Manzanares, A. Ba˜ nos, E. Guillamón, A.M. García-Campa˜ na, High-performance liquid chromatography method for the monitoring of the Allium derivative propyl propane thiosulfinate used as natural additive in animal feed, Food Anal. Methods 8 (2015) 916–921. [27] M. Llana-Ruiz-Cabello, S. Pichardo, N.T. Jiménez-Morillo, J.M. Bermúdez, S. Aucejo, F.J. González-Vila, A.M. Cameán, J.A. González-Pérez, Molecular Q5 characterization of a bio-based active package containing Origanum vulgare L. essential oil using analytical pyrolysis (Py-GC/MS), J. Sci. Food Agric. (2016), http://dx.doi.org/10.1002/jsfa.7502. [28] M. Llana-Ruíz-Cabello, S. Pichardo, N.T. Jimenez-Morillo, P. Abad, E. Guillamon, F.J. González-Vila, A.M. Cameán, J.A. Gonzalez-Perez, Characterization of a bio-based package containing a natural additive from Q6 Allium spp. using analytical pyrolysis and carbon stable isotopes, J. Anal. Appl. Pyrolysis (2016) (in press). [29] V. Lanzotti, The analysis of onion and garlic, J. Chromatogr. A 1112 (2006) 3–22. [30] L. Gîtin, R. Dinic, C. Neagu, L. Dumitrascu, Sulfur compounds identification and quantification from Allium spp. fresh leaves, J. Food Drug Anal. 22 (2014) 425–430. [31] N.C.J. Packia Lekshmi, S. Viveka, M.B. Viswanathan, T. Mini Shobi, GC–MS Characterization of organic sulphur compounds and other volatile odorous compounds from Allium sativum, Asian J. Microbiol. Biotechnol. Environ. Sci. 17 (2015) 73–78. [32] J.A. Gonzalez-Perez, G. Almendros, J.M. de la Rosa, F.J. Gonzalez-Vila, Appraisal of polycyclic aromatic hydrocarbons (PAHs) in environmental matrices by analytical pyrolysis (Py-GC/MS), J. Anal. Appl. Pyrolysis 109 (2014) 1–8. [33] M. Llana-Ruiz-Cabello, S. Maisanaba, M. Puerto, A.I. Prieto, S. Pichardo, A. Jos, A.M. Cameán, Evaluation of the mutagenicity and genotoxic potential of carvacrol and thymol using the Ames Salmonella test and alkaline, Endo III and FPG-modified comet assays with the human cell line Caco-2, Food Chem. Toxicol. 72 (2014) 122–128. [34] S. Shaposhnikov, A. Azqueta, S. Henriksson, S. Meier, I. Gaivao, N.H. Huskisson, A. Smart, G. Brunborg, M. Nilsson, A.R. Collins, Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation, Toxicol. Lett. 195 (2010) 31–34. [35] S. Maisanaba, D. Gutiérrez-Praena, M. Puerto, M. Llana-Ruiz-Cabello, S. Pichardo, R. Moyano, A. Blanco, M. Jordá, A. Jos, In vivo toxicity evaluation of the migration extract of an organomodified clay–poly(lactic) acid nanocomposite, J. Toxicol. Environ. Health Part A 77 (2014) 731–746. [36] M. Llana-Ruiz-Cabello, S. Pichardo, S. Maisanaba, M. Puerto, A.I. Prieto, D. Gutiérrez-Praena, A. Jos, A.M. Cameán, In vitro toxicological evaluation of essential oils and their main compounds used in active food packaging: a review, Food Chem. Toxicol. 81 (2015) 9–27. [37] P. Fan, H. Lou, Effects of polyphenols from grape seeds on oxidative damage to cellular DNBA, Mol. Cell. Biochem. 267 (2004) 67–74. [38] F. Bakkali, S. Averbeck, D. Averbeck, M. Idaomar, Biological effects of essential oils—a review, Food Chem. Toxicol. 46 (2008) 446–475. [39] F.M. Escobar, L.N. Cariddi, M.C. Sabini, E. Reinoso, S.B. Sutil, C.V. Torres, S.M. Zanon, L.I. Sabini, Lack of cytotoxic and genotoxic effects of Minthostachys verticillata essential oil: studies in vitro and in vivo, Food Chem. Toxicol. 50 (2012) 3062–3067. 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 Please cite this article in press as: P. Mellado-García, et al., Genotoxicity of a thiosulfonate compound derived from Allium sp. intended to be used in active food packaging: In vivo comet assay and micronucleus test, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2016), http://dx.doi.org/10.1016/j.mrgentox.2016.03.001 ARTICLE IN PRESS G Model MUTGEN 402712 1–11 P. Mellado-García et al. / Mutation Research xxx (2016) xxx–xxx 11 [40] V.B. Liju, K. Jeena, R. Kuttan, Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric (Curcuma longa L.), Food Chem. Toxicol. 53 (2013) 52–61. [41] Y. Shukla, N. Kalra, Cancer chemoprevention with garlic and its constituents, Cancer Lett. 247 (2007) 167–181. [42] Y. Shukla, P. Taneja, Antimutagenic effects of garlic on chromosomal aberrations, Cancer Lett. 176 (2002) 31–36. [43] M.Y. Shon, S.D. Choi, G.G. Kahng, S.H. Nam, N.K. Sung, Antimutagenic antioxidant and free radical scavenging activity of ethyl acetate extracts from white, yellow and red onions, Food Chem. Toxicol. 42 (2004) 659–666. [44] D. Guyonnet, C. Belloir, M. Suschetet, M.H. Siess, A.M. Le bon, Liver subcellular fractions from rats treated by organosulfur compounds from Allium modulate mutagen activation, Mutat. Res. 466 (2000) 17–26. [45] D. Guyonnet, C. Belloir, M. Suschetet, M.H. Siess, A.M. Le bon, Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction, Mutat. Res. 495 (2001) 135–145. [46] A. García, A.I. Haza, N. Arranza, M.E. Delgado, J. Rafter, P. Morales, Organosulfur compounds alone or in combination with vitamin C protect towards N-nitrosopiperidine- and N-nitrosodibutylamine-induced oxidative DNA damage in HepG2 cells, Chem. Biol. Interact. 173 (2008) 9–18. [47] C. Belloir, V. Singh, C. Daurat, M.H. Siess, A.M. Le bon, Protective effects of garlic sulfur compounds against DNA damage induced by direct- and indirect-acting genotoxic agents in HepG2 cells, Food Chem. Toxicol. 44 (2006) 827–834. [48] S.R.R. Musk, P. Clapham, I.T. Johnson, Cytotoxicity and genotoxicity of diallyl sulfide and diallyl disulfide towards Chinese hamster ovary cells, Food Chem. Toxicol. 35 (1997) 379–385. [49] D. Kirkland, S. Pfuhler, D. Tweats, M. Aardema, R. Corvi, F. Darroudi, A. Elhajouji, H. Glatt, et al., How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: report of an ECVAM Workshop, Mutat. Res. 628 (2007) 31–55. [50] S.H. Kang, N.J.Y. Kwo, J.K. Lee, Y.R. Seo, Recent advances in in vivo genotoxicity testing: prediction of carcinogenic potential using comet and micronucleus assay in animal models, J. Cancer Prev. 4 (2013) 277–288. [51] A.R. Collins, Measuring oxidative damage to DNA and its repair with the comet assay, Biochim. Biophys. Acta 1840 (2014) 794–800. [52] M.A. Alnaqee, M. Thomson, T. Bordia, M. Ali, Histopathological effects of garlic on liver and lung of rats, Toxicol. Lett. 85 (1996) 157–164. [53] S.K. Banerjee, M. Maulik, S.C. Manchanda, A.K. Dinda, T.K. Das, S.K. Maulik, Garlic-induced alteration in rat liver and kidney morphology and associated changes in endogenous antioxidant status, Food Chem. Toxicol. 39 (2001) 793–797. [54] S.K. Banerjee, M. Maulik, S.C. Manchanda, A.K. Dinda, S.K. Gupta, S.K. Maulik, Dose-dependent induction of endogenous antioxidants in rat heart by chronic administration of garlic, Life Sci. 70 (2002) 1509–1518. [55] EFSA Scientific Committee, Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment, EFSA J. 9 (2011) 2379. [56] S. Barlow, Threshold of Toxicological Concern (TTC), A tool for assessing substances of unknown toxicity present at low levels in the diet. ILSI Concise Monograph Series, ILSI Press, Brussels, 2005. [57] EFSA, Scientific opinion on xxploring options for providing advice about possible human health risks based on the concept of Threshold of Toxicological Concern (TTC), EFSA J. 10 (2012) 2750. 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 103 Capítulo III / Chapter III P. Mellado-García, M. Puerto, S. Pichardo, M. Llana-Ruiz-Cabello, R. Moyano, A. Blanco, A. Jos, A.M. Cameán TOXICOLOGICAL EVALUATION OF AN ALLIUM-BASED COMMERCIAL PRODUCT IN A 90-DAY FEEDING STUDY IN SPRAGUE–DAWLEY RATS Food and Chemical Toxicology 90, 18–29, 2016 104 Dear Author, Please, note that changes made to the HTML content will be added to the article before publication, but are not reflected in this PDF. Note also that this file should not be used for submitting corrections. Toxicological evaluation of an Allium-based commercial product in a 90-day feeding study in SpragueeDawley rats Q6 Q5 P. Mellado-García a , M. Puerto a , S. Pichardo a , M. Llana-Ruiz-Cabello a , R. Moyano b , A. Blanco c ,A.Jos a , A.M. Came an a , * a Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García Gonz alez n  2, 41012, Seville, Spain b Department of Pharmacology, Toxicology and Legal and Forensic Medicine, University of C ordoba, Campus de Rabanales Carretera Madrid-C adiz s/n, C ordoba, 14071, Spain c Department of Anatomy and Comparative Pathology and Anatomy, University of C ordoba, Campus de Rabanales Carretera Madrid-C adiz s/n, C ordoba, 14071, Spain article info Article history: Received 14 December 2015 Received in revised form 8 January 2016 Accepted 25 January 2016 Available online xxx Keywords: Allium extract Proallium AP ® Subchronic toxicity 90-Day oral Rats abstract Proallium AP ® is a commercial Allium extract intended to be used in active food packaging as the antibacterial and antioxidant effects of some organosulfur compounds are well known. However, there is little information on its toxicity and the Scientific Committee on Food (UE) requires the safety assessment of substances used in food contact materials. Thus, the aim of this study was to conduct for the first time a subchronic oral toxicity study of Proallium AP ® with groups of 10 males and 10 females Sprague eDawley rats fed a diet containing 0, 25, 100, 400 mg/kg/d for 90 days. No treatment-related clinical signs or mortality were noted. Besides, no treatment-related effects with regard to any of the toxicological biomarkers considered were observed, including biochemical, haematological and histopathology parameters. In conclusion, the non-observed-adverse-effect-level (NOAEL) for Proallium AP ® in rats was determined to be a dietary dose of 400 mg/kg/d under the present experimental conditions, a value 500- fold higher than the exposure derived from its potential use in active packaging. ©2016 Published by Elsevier Ltd. 1. Introduction Scientific research has focused its interest on essential oils (EOs) from medicinal plants as natural sources of antimicrobial agents and antioxidants (Hassanien et al., 2015). EOs vary in odor and flavor, which are governed by the types and amount of their constituents (Tongnuanchan and Benjakul, 2014). Allium cepa (onion) and Allium sativum (garlic) exhibit marked antibacterial activity and antioxidant effects (Benkeblia, 2004; Ye et al., 2013); moreover, both of them are plant species containing organosulfur compounds (OSCs) with applications in active food packaging (Llana-Ruiz- Cabello et al., 2015a). Active food contact materials were defined in Regulation No 1935/2004 of the Europe Parliament and of the Council as “materials that are intended to extend the shelf-life or to maintain or improve the condition of packaged food. They are designed to deliberately incorporate components that would release or absorb substances into or from the packaged food or the environment surrounding the food”. The nature of active agents that can be incorporated is very diverse and includes organic acids, enzymes, bacteriocins, fungicides, natural extracts, ions and ethanol as well as the materials in which they are included, e.g., papers, plastics, metals or mixture of these materials (Danielli et al., 2008). Besides, more research is needed to develop cheaper, more easily applicable and effective packaging systems (Lee et al., 2015). Because of petroleum-based matrices used to develop food contact material take hundreds of years to decompose, biodegradable materials based on starch, such as polylactic acid (PLA) has attracted the attention of the industry (Debiagi et al., 2014). Thus, active films have been developed by extrusion using PLA incorporated with natural substances as antimicrobial compounds (Del Nobile et al., 2009). In this sense, recently, the use of PLA films containing different percentages of an Allium spp extract (2%, 5% and 6.5% Proallium AP ® ) to be employed in the packaging of ready-to-eat salads have been proposed by our group (Llana-Ruiz-Cabello et al., 2015b). The developed films, especially those containing 5% and 6.5% Proallium AP ® exhibit strong antimicrobial effects. Proallium AP ® is a *Corresponding author. Q 1 E-mail address: [email protected] (A.M. Came an). Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox http://dx.doi.org/10.1016/j.fct.2016.01.019 0278-6915/©2016 Published by Elsevier Ltd. Food and Chemical Toxicology xxx (2016) 1e12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 FCT8496_proof ■28 January 2016 ■1/12 Please cite this article in press as: Mellado-García, P., et al., Toxicological evaluation of an Allium-based commercial product in a 90-day feeding study in SpragueeDawley rats, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.01.019 commercial product based on lipid-soluble OSCs, which are characteristic of the plant genus Allium spp, and it is used as flavoring of sauces, prepared meals and salads (Llana-Ruiz-Cabello et al., 2015b). Propyl thiosulphinate oxide (PTSO) is one of these OSCs obtained by the decomposition of initial components present in Allium spp. and it is incorporated in an inert commercial food-grade support (cyclodextrin) to produce Proallium AP ® . The antimicrobial properties and the antioxidant role of PTSO have been previously demonstrated (Llana-Ruiz-Cabello et al., 2015c; Peinado et al., 2012, 2013; Ruiz et al., 2010). Other minoritary components of this Allium extract are dipropylsulfiide (DPS) and dipropyl disulfide (DPDS) (Llana-Ruiz-Cabello et al., 2015d). The incorporation of EOs in active food packaging can result in a higher human exposure and consequently, more research is needed to establish effective and safe concentrations of EOs (Seow et al., 2014). In the case of active packaging, the EOs or natural extracts allowed to be used for this purpose have been not published so far in Europe. The Guidelines of the Scientific Committee on Food for safety assessment of substances used in food contact materials (European Commission, 2001), which have been recently updated (EFSA, 2008, 2015), establish the toxicological tests that have to be supplied for substances intended to be used in food contact materials. Thus, the core set includes at least 3 mutagenicity in vitro studies and a 90-day oral toxicity study in rodents. As far as we know, no components of Allium EO have been authorised for their use as active agent in food packaging, and their safety needs to be confirmed before their use in the food industry. After reviewing the scientific literature, in the case of OSCs of garlic/onion extracts, few toxicological studies have been described. The minor components, DPS and DPDS did not show any cytototoxic effects in Caco-2 cells (0e200 m M) nor mutagenic effects in the Ames test in the concentration range 0.1e200 m M(Llana-Ruiz-Cabello et al., 2015d). Moreover, Musk et al. (1997) observed significant changes in the number of chromosomal aberrations (CA), as well as in the sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells after diallyl sulfide (DAS) and diallyl disulfide (DADS) exposure. In relation to PTSO, the major OSC present in Proallium ® ,in vitro cytototoxic effects have been described in Caco-2 cells and HepG2 cells in a range between 350 and 415 m M, being 10-fold higher than the concentrations used in food packaging (Llana-Ruiz-Cabello et al., 2015c). Moreover, the potential mutagenicity/genotoxicity of PTSO has been recently assessed in vitro by a battery of genotoxicity tests at relevant concentrations according to its use in food packaging (0e50 m M). This compound exhibited a weak mutagenic potential on L5178YTk þ/- cells after 24 h of treatment using the mouse-lymphoma assay (MLA), and an increase of binucleated cells with micronuclei (BNMN) frequency in presence of metabolic activation S9 (20 m M) (Mellado-García et al., 2015). With respect to in vivo experiments, Guyonnet et al. (2000) have demonstrated the effects of some OSCs (DAS, DADS, DPS and DPDS) on the activation of several mutagens in male Wistar rats, and various results were reported depending on the mutagen considered in each case. A preliminary in vivo acute study of PTSO has been conducted in rats orally exposed by gavage to establish the maximum tolerated dose (MTD) of this compound (Llana-Ruiz-Cabello et al., 2015c). In relation to in vivo 90-day oral toxicity studies in rats of EOs with application in food industry (functional food ingredients, food packaging, etc.), only a few of them have been performed to assess their safety, such as: the ethanolic extract of Artemisia dracunculus L. (Ribnicky et al., 2004), the turmeric EO (Curcuma longa)(Liju et al., 2013), the ginger oil (Zingiber officinale)(Jeena et al., 2014), the peperina oil (Minthostachys verticillata)(Escobar et al., 2014), an Fig. 1. Mean body weights of a) male and b) female rats exposed to Proallium AP ® (25, 100 and 400 mg/kg/d) by oral route and control rats for 90 days. Table 1 Performance of SpragueeDawley rats fed with different doses of Proallium AP ® in the diet for 90-day. Values represent the mean ±SD of 10 rats/sex/group. Differences between control and treated groups for male and female rats were evaluated by KruskaleWallis test (K.W.) or by ANOVA test (F values). Parameters Male Female Group 1 (0 mg/ kg/day) Group 2 (25 mg/ kg/day) Group 3 (100 mg/ kg/day) Group 4 (400 mg/ kg/day) Group 1 (0 mg/ kg/day) Group 2 (25 mg/ kg/day) Group 3 (100 mg/ kg/day) Group 4 (400 mg/ kg/day) N¼10 N ¼10 N ¼10 N ¼10 N ¼10 N ¼10 N ¼10 N ¼10 Initial body weight (g) 268.3 ±14.2 265.5 ±13.4 266.4 ±15.6 267.3 ±18.0 185 ±12.8 192.1 ±8.1 190.1 ±13.2 191 ±10.6 F(36.3) ¼0.5511 p ¼0.9827; N.S. F(36.3) ¼0.7762 p ¼0.5150; N.S. Final body weight (g) 492 ±25.3 518.5 ±50.6 515.1 ±57.2 508.1 ±54.8 291 ±17.9 291.6 ±20.5 287.4 ±25.5 293.1 ±21.0 F(36.3) ¼0.5856 p ¼0.6284; N.S. F(36.3)¼0.1274 p ¼0.9433; N.S. Body weight gain 223.7 ±19.27 253.0 ±43.97 248.7 ±48.60 240.8 ±40.11 106.0 ±16.03 99.5 ±14.11 97.7 ±17.49 102.1 ±14.00 KW ¼2.028 p ¼0.5666; N.S. F(36.3)¼0.5311 p ¼0.6639; N.S. Total feed intake (g) 2094.6 ±177.3 2194.4 ±186.4 2112.6 ±262.0 2137.9 ±152.9 1491.2 ±67.4 1531.9 ±107.7 1501.3 ±169.1 1549.3 ±98.6 F(36.3) ¼1.065 p ¼0.3759; N.S. KW ¼1.587 p ¼0.6622; N.S. Feed conversion ratio 9.37 ±0.66 8.82 ±0.94 8,63 ±0,93 9.01 ±1.00 14.40 ±2.57 15.64 ±2.16 15.66 ±1.86 15.36 ±1.71 F(36.3)¼1.315 p ¼0.2846; N.S. F(36.3) ¼0.8005 p ¼0.5018; N.S. N.S.: Not Significant. P. Mellado-García et al. / Food and Chemical Toxicology xxx (2016) 1e122 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FCT8496_proof ■28 January 2016 ■2/12 Please cite this article in press as: Mellado-García, P., et al., Toxicological evaluation of an Allium-based commercial product in a 90-day feeding study in SpragueeDawley rats, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.01.019 extract of Sophorae radix (Che et al., 2015), and an aqueous dry spearmint extract (Lasrado et al., 2015). However, to the extend of our knowledge, specifically dose repeated studies that examine the safety of Allium extracts, their components, or Proallium AP ® are non-existent. Taking into account these facts, in the present study we have assessed, for the first time, the toxicity of Proallium AP ® intended to be used in active food packaging through a 90-day subchronic toxicity study in male and female SpragueeDawley rats according to the principles of the OECD 408 guideline (TG) (OECD, 1998). We have looked into several parameters including body weight changes, food and water consumption, feed conversion efficiency, haematological and biochemistry parameters, organ weights after necropsy and histopathology of various tissues. The results would allow to obtain an estimation of a non-observed-adverse-effect level (NOAEL) of exposure to establish safety criteria for human exposure to this extract. 2. Materials and methods 2.1. Test substance Proallium AP ® , a commercial Allium spp. preparation used in this study was provided by DOMCA S.A. (Granada, Spain). The concentration of the main OSCs, PTSO in the product was 14.5%. Commercial powder neutral gelatine from pork protein (Jesus Navarro S.A., Alicante, Spain) was used as the vehicle for the test item in the dosed groups and control group. For the 90-day study, dietary dose individual formulations were prepared daily for the whole experiment (13 weeks), and on fridays they were also prepared for the weekend. For dietary dose individual formulation, the test substance was included in 3 mL neutral gelatine (from a solution prepared with12 slides/100 mL warm water). The quantities of the test substance added ( m L of Proallium AP ® ) depended on the dose selected for each experimental group, and were solidified at 4  C, and stored at this temperature overnight, protected from light. Homogeneity of the dietary dose formulations and their stability were confirmed to be at least 5 days. 2.2. Animals and husbandry Forty male and forty female rats of SpragueeDawley strain (CRL:OFA (SD)) were supplied by Charles River Laboratories S.L. (Kings, NY, USA), and they were received at approximately 7 weeks of age and stabilized for at least 7 days. During acclimatization, the animals were examined by a veterinary surgeon. At the start of dosing, at approximately 8 weeks of age, average body weight of the males was 267.0 g (range 245e301 g) and of the females was 189.5 g (166e209 g). Animals were individually housed in cages type 3H with Souralit Fig. 2. Mean body weight gain (%) of a) male and b) female rats exposed to Proallium AP ® (25, 100 and 400 mg/kg/d) by oral route and control rats for 90-day. Fig. 3. Food consumption pattern (g) of a) male and b) female rats exposed to Proallium AP ® (25, 100 and 400 mg/kg/d) by oral route and control rats for 90-day. Fig. 4. Water consumption pattern (g) of a) male and b) female rats exposed to Proallium AP ® (25, 100 and 400 mg/kg/d) by oral route and control rats for 90-day. P. Mellado-García et al. / Food and Chemical Toxicology xxx (2016) 1e12 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FCT8496_proof ■28 January 2016 ■3/12 Please cite this article in press as: Mellado-García, P., et al., Toxicological evaluation of an Allium-based commercial product in a 90-day feeding study in SpragueeDawley rats, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.01.019 on Food for safety assessment of substances used in food contact materials (European Commission, 2001), in vivo toxicological studies are needed to establish effective and safe concentrations, such as a 90-day oral toxicity study in rodents. The OECD guideline 408 (1998) provides information on the possible health hazards likely to arise from repeated exposure over a prolonged period of Fig. 5. Histopathological examination of liver, kidney, intestines and stomach of male (A) and female (C) untreated (control) SpragueeDawley rats, and male (B) and female (D) rats subchronically exposed to 400 mg/kg b.w./d Proallium AP ® for 90-day. Representative sections of these organs (50 m m). In liver, normal hepatic cords and normal polyhedral hepatocytes (He) appeared with central nucleus and clear cytoplasm in all cases. In the kidney, renal parenchyma with normal glomeruli (GI) and renal tubules in male and female control rats (A,C), which are conserved in treated rats with the highest dose of Proallium AP ® administered (B,D) are shown. Intestinal villi (Vi) with abundant apparently normal enterocytes (En) were observed in all cases (AeD). Detail of the glandular stomach with mucosal (Mc) and glandular (Gc) cells apparently normal in control and treated rats (AeD). Fig. 6. Histopathological examination of lung, heart and testicule/ovary of male (A) and female (C) untreated (control) SpragueeDawley rats, and male (B) and female (D) rats subchronically exposed to 400 mg/kg b.w./d Proallium AP ® for 90-day. Representative sections of these organs (50 m m). Detail of control bronchial epithelia, without any alterations in bronchia (Br) and alveoli (Al) in untreated rats (A,C) which were conserved in treated rats with the highest dose of 400 mg/kg b.w./d of Proallium AP ® . In heart, normal cardiac fibers (Fi) were observed in all cases (AeD). Testicles of male rats showed normal seminiferous tubules (St) and interstitial space (Is) (A) which was maintained in treated male rats (B); Ovaries from control and treated female rats (C,D) presented normal follicles (F) in all cases. P. Mellado-García et al. / Food and Chemical Toxicology xxx (2016) 1e1210 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FCT8496_proof ■28 January 2016 ■10/12 Please cite this article in press as: Mellado-García, P., et al., Toxicological evaluation of an Allium-based commercial product in a 90-day feeding study in SpragueeDawley rats, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.01.019 time covering post-weaning maturation and growth well into adulthood. In vivo toxicological information of Proallium AP ® was not available in the scientific literature, and only an acute study of its main compound PTSO had been carried out (Llana-Ruiz-Cabello et al., 2015c). In this work, we have performed for the first time a 90-day oral toxicity study in male and female rats, after administration of the Proallium AP ® preparation. Overall, the doses assayed (0e400 mg/kg/d) did not produce any significant adverse change in any of the parameters considered. These results agree with the negative pathological changes detected in the histopathological analysis. Therefore, Proallium AP ® appears to be safe and non-toxic in the study performed and a NOAEL in rats is established at the highest dose assayed of 400 mg/kg/day. In this study, the main component of Proallium AP ® essential oil was PTSO, representing 14.5% of the total oil (Llana-Ruiz-Cabello et al., 2015c). In relation to PTSO, a previous acute in vivo toxicity study in rats orally exposed by gavage to 2000 and 175 mg/kg b.w. revealed macroscopically that this compound induced inflammation in the intestine and liver of rats. Similarly, light microscopy revealed damage at those doses, showing degenerative processes in hepatic parenchyma with loss of structure and hepatocytes in necrotic process at 2000 mg/kg b.w., and minor alterations in hepatic parenchyma were also observed at 175 mg/kg b.w. Moreover, desquamation processes were observed in the intestinal mucosal epithelium of rats at both doses assayed, whereas no remarkable morphological changes were detected in rats gavaged with 55 mg/ kg bw which was set as MTD. The induction of these toxic acute effects of PTSO in rats by gavage contrast with the negative histopathological findings reported in this work after repeated oral administration of doses of Proallium AP ® (up to 400 mg/kg/d) for 90 days. These differences could be due to the nature and composition of the substances assayed, PTSO versus the Allium extract indicating a higher safety of the extract to be used in food packaging. Some authors indicated that EOs have other constituents interacting as a whole with synergistic/antagonistic phenomena (Escobar et al., 2014; Pavlidou et al., 2004). Other factors should be taken into account, such as the route of administration, mixed with the diet in this work versus gavage in the PTSO study. In comparison to subchronic studies carried out with other EOs, recent reports informed that ginger (Z. officinale)(Jeena et al., 2014), turmeric (C. longa L.) (Liju et al., 2013) and peperina (M. verticillata) (Escobar et al., 2014) Q2 EOs are safe in rats up to an oral dose of 500 mg/kg b.w./day, similarly to the results obtained in this study. Only in the case of peperina EO was there an increase in weight gain and FCE in rats fed with 1 g/kg food, although it did not follow a doseeresponse pattern. The authors indicated that this EO may influence the palatability of food, and several researchers have demonstrated the EOs can be used as food additives because they improved the productive parameters of farmyard animals (Li et al., 2012; Yan et al., 2010). This fact has not been demonstrated in this work. The NOAEL found in this in vivo study of 400 mg/kg/day of Proallium AP ® is of high importance considering its safety to be used in active packaging. Thus, in the worst exposure scenario, for an adult consumer weighting 60 kg, the dose of 400 mg/kg/day corresponds to an ingestion of 24,000 mg of Proallium AP ® per day, which is 500-fold higher than this maximum potential exposure, so no potential subchronic toxic effects associated to the consumption of Proallium AP ® derived from its use in active packaging could be expected, according to the experimental conditions assayed. 5. Conclusion The present study reports for the first time that after a 90-day repeated dose oral exposure to Proallium AP ® at doses of 25, 100 and 400 mg/kg b.w./day, male and female rats did not show any changing trends on individual body weight, relative gain weight, feed conversion efficiency, food and water consumption, as well as on biochemical and haematological parameters. Moreover, there were no statistically significant differences between treated and control rats for terminal body weight or any absolute or relative weighed organs, and no histopathological abnormalities were observed associated with the treatment. Therefore, Proallium AP ® appears to be safe after subchronic administration and a NOAEL in rats is established at 400 mg/kg/day under the test conditions employed, a value 500-fold higher than the exposure derived from its potential use in active packaging. Acknowledgments The authors wish to thank the Spanish Ministry of Science and Innovation (AGL2012-38357-C02-01), co-financed with FEDER funds, and the Junta de Andalucía (AGR-7252) for the financial support of this study. María Llana-Ruiz-Cabello also wishes to thank the Junta de Andalucía for her predoctoral grant associated to AGR- 7252. Q3 Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fct.2016.01.019. Uncited reference OECD, 2008. Q4 References Benkeblia, N., 2004. Antimicrobial activity of essential oil extracts of various onions (Allium cepa) and garlic (Allium sativum). Food. Sci. Technol. 37, 263e268. Che, J.H., Yun, J.W., Kim, Y.S., Kim, S.H., You, J.R., Jang, J.J., Kim, H.C., Kim, H.K., Kang, B.C., 2015. Genotoxicity and subchronic toxicity of dophorae radix in rats: hepatotoxic and genotoxic potential. Regul. Toxicol. Pharmacol. 71, 379e387. Danielli, D., Gontard, N., Spyropoulos, D., Zondervan-van den Beuken, E., Tobback, P., 2008. Active and intelligent food packaging: legal aspects and safety concerns. Trends Food Sci. Technol. 19, S103eS112. Debiagi, F., Kobayashi, R.K.T., Nakazato, G., Panagio, L.A., Mali, S., 2014. Biodegradable active packaging base don cassava bagasse, polyvinyl alcohol and essential oils. Ind. Crop. Prod. 52, 664e670. Decision 2012/707/UE. Commission Implementing Decision of 14 November 2012 establishing a common format for the submission of the information pursuant to Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes <http://ec.europa. eu/environment/chemicals/lab_animals/pdf/Unofficial%20Consolidated% 20version.pdf [last access 11/12/2015]. Del Nobile, M.A., Conte, A., Buonocore, G.G., Incoronato, A.L., Massaro, A.M., Oanza, O., 2009. Active packaging by extrusion processing of recyclable and biodegradable polymers. J. Food Eng. 93, 1e5. Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes. <http://eur-lex.europa.eu/legalcontent/EN/TXT/?uri¼celex:32010L0063 [last access 11/12/2015]. Escobar, F.M., Sabini, M.C., Cariddi, L.N., Sabini, L.I., Ma~ nas, F., Cristofolini, A., Bagnis, G., Gallucci, M.N., Cavaglieri, L.R., 2014. Safety assessment of essential oil from Minthostachys verticillata (Griseb.) Epling (peperina): 90-days oral subchronic toxicity study in rats. Regul. Toxicol. Pharm. 71, 1e7. European Commission, 2001. Guidelines of the Scientific Committee on Food for the Presentation of an Application for Safety Assessment of a Substance to Be Used in Food Contact Materials Prior to its Authorization, pp. 1e6. SCF/CS/PLEN/GEN/ 100 Final 19 December 2001. European Food Safety Authority, 2008. Guidance Document on the Submission of a Dossier on a Substance to Be Used in Food Contact Materials for Evaluation by EFSA by the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) [Last access 11.12.15]. http://www.efsa.europa.eu/ en/efsajournal/doc/21r.pdf. European Food Safety Authority CEF Panel, (EFSA Panel on Food Contact Materials, Enzymes, Flavourings 49 and Processing Aids), 2015. Draft Scientific Opinion on Recent Developments in the Risk Assessment of Chemicals in Food and Their Potential Impact on the Safety Assessment of Substances Used in Food Contact Materials, pp. 1e22. http://www.efsa.europa.eu/sites/default/files/150707.pdf [last access 11.12.15]. P. Mellado-García et al. / Food and Chemical Toxicology xxx (2016) 1e12 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 FCT8496_proof ■28 January 2016 ■11/12 Please cite this article in press as: Mellado-García, P., et al., Toxicological evaluation of an Allium-based commercial product in a 90-day feeding study in SpragueeDawley rats, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.01.019 Guyonnet, D., Belloir, C., Suschetet, M., Siess, M.H., Le Bon, A.M., 2000. Liver subcellular fractions from rats treated by organosulfur compounds from Allium modulate mutagen activation. Mutat. Res. 466, 17e26. Hassanien, M.F., Assiri, A.M., Alzohairy, A.M., Oraby, H.F., 2015. Health-promoting value and food applications of black cumin essential oil: an overview. J. Food. Sci. Technol. 52, 6136e6142. Jeena, K., Liju, V.B., Kuttan, R., 2014. A preliminary 13-week oral toxicity study of ginger oil in male and female wistar rats. Int. J. Toxicol. 30, 662e670. Lasrado, J.A., Trinker, D., Ceddia, M.A., Herrlinger, K.A., 2015. The safety of a dry spearmint extract in vitro and in vivo. Regul. Toxicol. Pharm. 71, 213e224. Lee, S.Y., Lee, S.J., Choi, D.S., Hur, S.J., 2015. Current topics in active and intelligent food packaging for preservation of fresh foods. J. Sci. Food. Agric. 95, 2799e2810. Li, S.Y., Ru, Y.J., Liu, M., Xu, B., P eron, A., Shi, X.G., 2012. The effect of essential oils on performance, immunity and gut microbial population in weaner pigs. Livest. Sci. 145, 119e123. Liju, V.B., Jeena, K., Kuttan, R., 2013. Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric (Curcuma longa L). Food. Chem. Toxicol. 53, 52e61. Llana-Ruiz-Cabello, M., Pichardo, S., Maisanaba, S., Puerto, M., Prieto, A.I., Gutierrez- Praena, D., Jos, D., Camean, A.M., 2015a. In vitro toxicological evaluation of essential oils and their main compounds used in active food packaging: a review. Food Chem. Toxicol. 81, 9e27. Llana-Ruiz-Cabello, S., Pichardo, A., Ba~ nos, C., Nú~ nez, J.M., Bermúdez, E., Guillam on, S., Aucejo, Came an, A.M., 2015b. Characterisation and evaluation of PLA films containing an extract of Allium spp. to be used in the packaging of ready-to-eat salads under controlled atmospheres. LWT Food. Sci. Technol. 64, 1354e1361. Llana-Ruiz-Cabello, M., Guti errez-Praena, D., Puerto, M., Pichardo, S., Moreno, F.J., Ba~ nos, A., Nu~ nez, C., Guillam on, E., Came an, A.M., 2015c. Acute toxicological studies of the main organosulfur compound derived from Allium sp. intended to be used in active food packaging. Food. Chem. Toxicol. 82, 1e11. Llana-Ruiz-Cabello, M., Maisanaba, S., Gutierrez-Praena, D., Prieto, A.I., Pichardo, S., Jos, A., Moreno, F.J., Camean, A.M., 2015d. Cytotoxic and mutagenic in vitro assessment of two organosulfur compounds derived from onion to be used in the food industry. Food. Chem. 166, 423e443. Mellado-García, P., Maisanaba, S., Puerto, M., Llana-Ruiz-Cabello, M., Prieto, A.I., Marcos, R., Pichardo, S., Came an, A.M., 2015. Genotoxicity assessment of propyl thiosulphinate oxide, an organosulfur compound from Allium extract, intended to food active packaging. Food. Chem. Toxicol. 86, 365e373. Musk, S.R.R., Clapham, P., Johnson, I.T., 1997. Cytoxicity and genotoxicity of diallyl sulfide and diallyl disulfide towards Chinese hamster ovary cells. Food. Chem. Toxicol. 35, 379e385. OECD (Organisation for Economic Cooperation and Development), 1998. Guideline for Testing of Chemicals. Repeated Dose 90-day Oral Toxicity Study in Rodents, Guideline 408, pp. 1e10. OECD (Organisation for Economic Cooperation and Development), 2008. Guidelines for the Testing of Chemicals. Acute Oral Toxicityup -and-down- Procedure (UDP). Guideline 425, pp. 1e27. Pavlidou, V., Karpouhtsis, I., Franzios, G., Zambetaki, A., Scouras, Z., Mavragani- Tsipidou, P., 2004. Insecticidal and genotoxic effects of essential oils of Greek sage, Salvia fruticosa, and mint, Mentha pulegium,onDrosophila melanogaster and Bactrocera oleae (Diptera: Tepritidae). J. Agr. Urban Entomol. 21, 39e49. Peinado, M.J., Ruiz, R., Ech avarri, A., Rubio, L.A., 2012. Garlic derivative propyl propane thiosulfonate is effective against broiler enteropathogens in vivo. Poult. Sci. 91, 2148e2157. Peinado, M.J., Ruiz, R., Ech avarri, A., Aranda-Olmedo, I., Rubio, L.A., 2013. Garlic derivative PTS-O modulates intestinal microbiota composition and improves digestibility in growing broiler chickens. Anim. Feed. Sci. Tech. 181, 87e92. RD 53/2013, 2013. Real Decreto, se establecen las normas b asicas aplicables para la protecci on de los animales utilizados en experimentaci on y otros fines científicos, incluyendo la docencia. BOE 34, 11370e11421. http://boe.es/boe/dias/ 2013/02/08/pdfs/BOE-A-2013-1337.pdf [last access 05.11.15]. Regulation (EC) No 1935/2004 of the European Parliament and of the Council of 27 October 2004 on materials and articles intended to come into contact with food and repealing Directives 80/590/EEC and 89/109/EEC. Ribnicky, D.M., Poulev, A., O'Neal, J., Wnorowski, G., Malek, D.E., J€ ager, R., Raskin, I., 2004. Toxicological evaluation of the ethanolic extract of Artemisia dracunculus L. for use as a dietary supplement and in functional foods. Food Chem. Toxicol. 42, 585e598. Ruiz, R., García, M.P., Lara, A., Rubio, L.A., 2010. Garlic derivatives (PTS and PTS-O) differently affect the ecology of swine faecal microbiota in vitro. Vet. Microbiol. 144, 110e117. Seow, Y.X., Yeo, C.R., Chung, H.L., Yuk, H.-G., 2014. Plant essential oils as active antimicrobial agents. Crit. Rev. Food Sci. Nutr. 54, 625e644. Tongnuanchan, P., Benjakul, S., 2014. Essential oils: extraction, bioactivities, and their uses for food preservation. J. Food. Sci. 79, 1231e1249. Yan, L., Wang, J.P., Kim, H.J., Meng, Q.W., Ao, X., Hong, S.M., Kim, I.H., 2010. Influence of essential oil supplementation and diets with different nutrient densities on growth performance, nutrient digestibility, blood characteristics, meat quality and fecal noxious gas content in growerefinisher pigs. Livest. Sci. 128, 115e122. Ye, C., Dai, L., Hu, W.L., 2013. Antimicrobial and antioxidant activities of the essential oil from onion (Allium cepa L.). Food Control. 30, 48e53. P. Mellado-García et al. / Food and Chemical Toxicology xxx (2016) 1e1212 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 FCT8496_proof ■28 January 2016 ■12/12 Please cite this article in press as: Mellado-García, P., et al., Toxicological evaluation of an Allium-based commercial product in a 90-day feeding study in SpragueeDawley rats, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.01.019 119 Capítulo IV / Chapter IV Pilar Mellado-García, Sara Maisanaba, María Puerto, Ana Isabel Prieto, Ricard Marcos, Silvia Pichardo, Ana María Cameán. IN VITRO TOXICOLOGICAL ASSESSMENT OF AN ORGANOSULFUR COMPOUND FROM ALLIUM EXTRACT: CYTOTOXICITY, MUTAGENICITY AND GENOTOXICITY STUDY Food and Chemical Toxicology 99, 231-240, 2017 120 Elsevier Editorial System(tm) for Food and Chemical Toxicology Manuscript Draft Manuscript Number: FCT-D-16-01816R1 Title: In vitro toxicological assessment of an organosulfur compound from Allium extract: cytotoxicity, mutagenicity and genotoxicity studies Article Type: Full Length Article Keywords: propyl propane thiosulfinate (PTS), Ames test, mouse lymphoma TK assay; micronucleus test; comet assay. Corresponding Author: Dr. Ana Cameán, Corresponding Author's Institution: First Author: Pilar Mellado-García Order of Authors: Pilar Mellado-García; Sara Maisanaba; Maria Puerto; Ana I Prieto; Ricard Marcos; Silvia Pichardo; Ana Cameán Abstract: Garlic (Allium sativum) and onion (Allium cepa) are being used in the food industry as flavoring but also for their antimicrobial activities.These activities are mainly derived from the organosulfur compounds (OSCs). Propyl propane thiosulfinate (PTS) is an OSC with potential use in the active packaging, but its safety should be guaranteed before being commercialized. The aim of this work was to investigate for the first time the cytotoxicity of PTS as well as its in vitro mutagenic/genotoxic potential using the following battery of genotoxicity tests:(1)the bacterial reverse-mutation assay in S. typhimurium (Ames test, OECD 471, 1997);(2) the micronucleus test (MN, OECD 487, 2016);(3) the mouse lymphoma thymidine-kinase assay (MLA, OECD 476, 2015), and (4)the comet assay (standard and modified with restriction enzymes). The results revealed that PTS was not mutagenic neither in the Ames test nor in MLA. However, genotoxic effects were recorded in the MN test on mammalian cells (L5178Y Tk+/-cells) after PTS exposure at the highest concentration tested (17.25 µM) without S9, and also its metabolites (+S9, from 20 µM). Moreover, in the comet assay, PTS induced DNA breaks damage in Caco-2 cells at the highest concentration tested (280 µM) but it did not induce oxidative DNA damage. Response to Reviewers: Reviewer #1: The article entitled "In vitro toxicological assessment of an organosulfur compound from Allium extract: cytotoxicity, mutagenicity and genotoxicity studies" describes am interesting work in which the toxicity of OSCs was evaluated. The article is well written and organized and deserves to be published after minor revision. In detail: Response: Thank you very much for your kind comments and for your revision. 1) OSCs are odorous compounds that produce a strong smell when they are used as additives and they go in contact with food. In this article, the authors speak about the use of these compounds in food active packaging applications. How the authors think to eliminate the problem of "smell transfering" to the packaged food in a system as active packaging (without afect the aroma of packaged food)? Response: The referee is right, OSCs are odorous compounds and this smell would be transfered to the packaging. In preliminary studies, we have evidenced that this smell in the film is not very strong but we cannot reduce the quantity of Allium extract in the film because it won't be effective. The highest concentration of active agent assed (6.5%) exhibited the best antimicrobial activity, being effective up to 5 days of storage for bacteria and even up to 7 days for moulds (Llana-Ruiz- Cabello et al., 2015. LWT - Food Science and Technology 64, 1354-1361). Nevertheless, these films are intended to be used in ready-to-eat salads when seasoning is desired. Our films area made by extruction, in case the smell of Allium wanted to be avoid, we would suggest to develop the active films by micro-encapsulation. 2) The quality of Figures 1 and 2 is not high, please improve it. Response: The quality of Figures 1 and 2 has been improved. Reviewer #3: The manuscript entitled "In vitro toxicological assessment of an organosulfur compound from Allium extract: cytotoxicity, mutagenicity and genotoxicity studies" is focused in the study of the toxicological profile of a new substance intended to be incorporated in food contact materials named as PTS. Several genotoxicity studies were performed and only positive results were observed in the micronucleus and in the standard comet assays. No toxicity was obtained in the Ames and mouse lymphoma tests. In general, the manuscript is well written, structured and complete. The topic of the manuscript is interesting and is in the scope of the Food and Chemical Toxicology. Since the present study is actually the first report on mutagenic and genotoxic activity of toxicological profile of PTS I suggest the publication of this manuscript doing minor changes before. There are only some minor comments to take into account: Response: Thank you very much for your kind comments and we are very grateful for your thoroughly revision of the manuscript. All your suggestions have been taken into account, and the manuscript has been improved. Abstract: - Write in italics "in vitro" and "S. typhimurium". Response: Sorry about the mistake. The changes have been made. - Unify the name of the cells L5178Y Tk+/- as is written through the manuscript. Response: Thank you. The cell name has been changed through the manuscript. Manuscript: - Introduction, page 1, line 52: Clarify that the OSCs are not present in a natural form, that they need to be incorporated. Rewrite as follow: "their use in food industry, particularly intended to be incorporated in FCM" Response: Thanks for your comment. The sentence has been changed according to your suggestion. - Introduction, page 2, line 31: Move the reference "EFSA, 2016" to the end of the sentence. Response: Thank you. The reference has been moved to the end of the sentence. - Introduction, page 2, line 38: The Guideline OECD 487 has been updated from July, 2016. Please change the reference through the manuscript from OECD 487, 2014 to OECD 487, 2016. Response: Thank you for your appreciation. The reference has been changed through the manuscript. - Introduction, page 3, line 29: Please, write also that cytotoxicity in L5178Y Tk+/- through the trypan blue exclusion test has been evaluated to complete the objectives. Response: Thank you for your appreciation. This information is already included in the manuscript. - M&M, page 3, line 59: Who provide the MTS? I think that there is a mistake in the format because in the text appeared "were purchased from...". Response: Sorry about the mistake, it is already corrected. - M&M, page 4, line 19: The cell reagents are the same for both cell cultures? Unify and avoid the repetitions. Response: Thank you for your comment. You are right, the cell reagents are the same in many cases. The sentence has been rewritten. - M&M, page 4, line 54: Include in this point 2.2. the bacterial strains used and delete from point 2.5. Response: Thank you very much. The bacterial strains have been specified in the point 2.2 and deleted from 2.5. - M&M, page 5, line 7: If it is possible move to their respective points the cell density to perform the assays, as in the other cases is done. Response: Thank you for your suggestion. The cell density to perform each assay has been written in each point. - M&M, page 5, line 33: Specify in point 2.4. which cell line is used in each assay. Response: Thanks. The cell lines used in each assay are already written. - Results, point 3.3, line 52: Unify the significant values (p<0.01 and p< 0.001) with the figure caption. Response: Thank you very much and sorry about the mistake. The change has been made. Dear Editor, Thank you very much for giving us an additional opportunity for the publication of our manuscript in Food and Chemical Toxicology. We are very grateful for the revision performed by all the reviewers and following their comments and all your suggestions we recognized that the manuscript has been improved. We hope that this final version will be accepted in your prestigious Journal. “In vitro toxicological assessment of an organosulfur compound from Allium extract: cytotoxicity, mutagenicity and genotoxicity studies” The authors of the article are: Pilar Mellado-García, Sara Maisanaba, María Puerto, Ana Isabel Prieto, Ricard Marcos, Silvia Pichardo, Ana María Cameán* I am looking forward to hearing from you as soon as possible Yours sincerely, Ana M. Cameán Corresponding author Cover Letter Please wait... If this message is not eventually replaced by the proper contents of the document, your PDF viewer may not be able to display this type of document. You can upgrade to the latest version of Adobe Reader for Windows®, Mac, or Linux® by visiting http://www.adobe.com/go/reader_download. For more assistance with Adobe Reader visit http://www.adobe.com/go/acrreader. Windows is either a registered trademark or a trademark of Microsoft Corporation in the United States and/or other countries. Mac is a trademark of Apple Inc., registered in the United States and other countries. Linux is the registered trademark of Linus Torvalds in the U.S. and other countries. *Conflict of Interest Statement In vitro toxicological assessment of an organosulfur compound from Allium extract: cytotoxicity, mutagenicity and genotoxicity studies Pilar Mellado-García1, Sara Maisanaba1, María Puerto1, Ana Isabel Prieto1, Ricard Marcos2, Silvia Pichardo1, Ana María Cameán1* 1Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n°2, 41012 Seville. Spain. 2Group of Mutagenesis, Department of Genetics and Microbiology, Universidad Autónoma of Barcelona, 08193 Cerdanyola del Valles, Barcelona, Spain. *Corresponding author: Ana M. Cameán Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n°2, 41012 Seville. Spain. E-mail address: [email protected] Tel: +34 954 556762 Fax: +34 954 556422 *Manuscript Title Page Highlights: - Genotoxic potential of PTS intended to use in active packaging - PTS was not mutagenic neither in the Ames test nor in MLA - PTS induces MN on mammalian cells (L5178Y Tk+/-cells) at the highest concentration tested, in presence or absence of S9 - PTS induced DNA breaks damage in Caco-2 cells at the highest concentration tested but it did not induce oxidative DNA damage. Highlights (for review) Abstract Garlic (Allium sativum) and onion (Allium cepa) are being used in the food industry as flavoring but also for their antimicrobial activities.These activities are mainly derived from the organosulfur compounds (OSCs). Propyl propane thiosulfinate (PTS) is an OSC with potential use in the active packaging, but its safety should be guaranteed before being commercialized. The aim of this work was to investigate for the first time the cytotoxicity of PTS as well as its in vitro mutagenic/genotoxic potential using the following battery of genotoxicity tests:(1)the bacterial reverse-mutation assay in S. typhimurium (Ames test, OECD 471, 1997);(2) the micronucleus test (MN, OECD 487, 2014);(3) the mouse lymphoma thymidine-kinase assay (MLA, OECD 476, 2015), and (4)the comet assay (standard and modified with restriction enzymes). The results revealed that PTS was not mutagenic neither in the Ames test nor in MLA. However, genotoxic effects were recorded in the MN test on mammalian cells (L5178Y Tk+/-cells) after PTS exposure at the highest concentration tested (17.25 µM) without S9, and also its metabolites (+S9, from 20 µM). Moreover, in the comet assay, PTS induced DNA breaks damage in Caco-2 cells at the highest concentration tested (280 µM) but it did not induce oxidative DNA damage. Keywords: propyl propane thiosulfinate (PTS), Ames test, mouse lymphoma TK assay; micronucleus test; comet assay. *Abstract In vitro toxicological assessment of an organosulfur compound from Allium extract: cytotoxicity, mutagenicity and genotoxicity studies Pilar Mellado-García1, Sara Maisanaba1, María Puerto1, Ana Isabel Prieto1, Ricard Marcos2, Silvia Pichardo1, Ana María Cameán1* 1Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n°2, 41012 Seville. Spain. 2Group of Mutagenesis, Department of Genetics and Microbiology, Universidad Autónoma of Barcelona, 08193 Cerdanyola del Valles, Barcelona, Spain. *Corresponding author: Ana M. Cameán Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n°2, 41012 Seville. Spain. E-mail address: [email protected] Tel: +34 954 556762 Fax: +34 954 556422 *Manuscript showing all track changes Abstract Garlic (Allium sativum) and onion (Allium cepa) are being used in the food industry as flavoring but also for their antimicrobial activities.These activities are mainly derived from the organosulfur compounds (OSCs). Propyl propane thiosulfinate (PTS) is an OSC with potential use in the active packaging, but its safety should be guaranteed before being commercialized. The aim of this work was to investigate for the first time the cytotoxicity of PTS as well as its in vitro mutagenic/genotoxic potential using the following battery of genotoxicity tests:(1)the bacterial reverse-mutation assay in S. typhimurium (Ames test, OECD 471, 1997);(2) the micronucleus test (MN, OECD 487, 20164);(3) the mouse lymphoma thymidine-kinase assay (MLA, OECD 476, 2015), and (4) the comet assay (standard and modified with restriction enzymes). The results revealed that PTS was not mutagenic neither in the Ames test nor in MLA. However, genotoxic effects were recorded in the MN test on mammalian cells (L5178Y Tk+/-cells) after PTS exposure at the highest concentration tested (17.25 µM) without S9, and also its metabolites (+S9, from 20 µM). Moreover, in the comet assay, PTS induced DNA breaks damage in Caco-2 cells at the highest concentration tested (280 µM) but it did not induce oxidative DNA damage. Keywords: propyl propane thiosulfinate (PTS), Ames test, mouse lymphoma TK assay; micronucleus test; comet assay. 1. Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... Formatted ... 2.1. Introduction Garlic, Allium sativum L., is considered one of the twenty most important vegetables with various uses throughout the world, not only for culinary purposes but also as an ingredient of traditional and modern medicine (Martins et al., 2016). In addition, onion, Allium cepa, has similar applications (Corzo-Martínez et al., 2007). They both exhibit a peculiar odour, which is associated with the presence of organosulfur compounds (OSCs), which are also responsible of their beneficial properties (Lekshmi et al., 2015). Essential oils (EOs) from garlic and onion have been studied for their use in the food industry (Mnayer et al., 2014). In fact, onion EO has been pointed out as a potential source of natural antimicrobial and antioxidant agents to be applied in food systems, due to its interesting properties (Benkeblia et al., 2004; Ye et al., 2013; Prakash et al., 2015). In this regard, the toxic assessment of extracts from garlic and onion, as well as their components, need to be addressed. The toxic assessment of OSCs is of great interest because their content can vary substantially depending on the different conditions (Benkeblia and Lanzotti, 2007). Previous works from our research group have studied the cytotoxicity and mutagenicity/genotoxicity of several OSCs such propyl propane thiosulfonate (PTSO) (Llana-Ruiz-Cabello et al. 2015a; Mellado-Garcia et al. 2015), dipropyl sulfide (DPS) and dipropyl disulfide (DPDS) (Llana-Ruiz-Cabello et al. 2015b). The obtained results evidenced a lack of toxic effects at the concentration ranges intended to be used for food packaging. Nevertheless other authors have reported both genotoxic (Musk et al., 1997) and antigenotoxic effects (Guyonnet et al., 2000, 2001; Belloir et al., 2006; Arranz et al., 2007; Chiu et al., 2016) for some OSCs. The contradictory results reported for these OSCs (LLana- Ruiz-Cabello et al., 2015c) made necessary to evaluate the toxicological profile of them before their use in food industry, particularly to intended to be incorporated in food contact materials (FCM). Despite the beneficial effects attributed to Allium plants, growing awareness exist on the hazards associated with the use of plants and their extracts as antibiotic and chemical feed additives (Wallace, 2004), as well as actives in food packaging (LLana-Ruiz-Cabello et al., 2015c; Maisanaba et al., 2016). Among the OSCs, propyl propane thiosulfinate (PTS) is an organosulfur compound obtained by decomposition of initial components present in Allium plants, that has been stabilized and characterized by DOMCA Research Center (DMC, Granada, Spain). This compound has previously shown to have beneficial effects on goats as methanogenesis inhibitor in the rumen (Martínez-Fernández et al., 2013, 2015). PTS is considered as a secondary metabolite of garlic, derived of the formation of the sulfenic acids, which nowadays has been studied for evaluate its antimicrobial activity against the faecal microbiota of pigs (Ruiz et al., 2015), or as dose-dependently killer of invasive sporozoites and stimulating of higher spleen cell proliferation in chickens (Kim et al., 2013). Nevertheless, little is known about its safety for human consumption. According to the Guidelines of the Scientific Committee on Food for Safety Assessment of Substances Used in FCM, information on the genotoxic potential is a key component in the risk assessment of substances used in active packaging (EFSA, 2011; 2016). In the new European Food Safety Authority (EFSA) Scientific Committee recommendations (EFSA, 2016) on genotoxicity testing strategies, a basic battery of two in vitro tests, a bacterial reverse mutation assay (OECD 471, 1997) and an in vitro mammalian cell micronucleus test (MN) (OECD 487, 20164) are recommended (EFSA, 2016). The bacterial reverse-mutation assay in Salmonella typhimurium (Ames test) is a short-term mutation study specifically designed to detect a wide range of chemical substances that can produce genetic damage leading to gene mutation. It is rapid and relatively easy to perform (Mortelmans and Zeiger, 2000). Concerning the MN test using mammalian cells, it is one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage, together with chromosome non-disjunction to be measured reliably (Fenech, 2000; EFSA, 2016). In the case of inconclusive, contradictory or positive results from these two in vitro tests, it may be appropriate to conduct further in vitro tests to optimize any subsequent in vivo testing, or to provide additional useful mechanistic information (EFSA, 2011, 2016). In this sense, the in vitro mammalian cell gene mutation assay (MLA) (OECD 476, 2015) is an assay commonly used, to evaluate the mutagenicity of chemical and physical agents This can detect a wide range of genetic alterations, including both point and chromosomal, gene, base pair substitutions and frame-shift mutations (Wang et al., 2009). Finally, the effect on DNA damage can be studied through the comet assay which is a reproducible and sensitive test for the detection of DNA damage (mainly DNA breaks) in eukaryotic cells. Incorporation of oxidative DNA damage repair enzymes (for example, formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (EndoIII)) in the standard alkaline comet assay procedure allows the detection and measurement of oxidatively DNA damaged (Collins, 2004; Pu et al., 2016). In the present work, a specifically toxicological approach has been carried out on PTS for the first time. The cytotoxicity of PTS was studied in the Caco-2 cell line by means of three endpoints: total protein content (TP), neutral red uptake (NRU) and MTS tetrazolium salt reduction, as well as in L5178Y Tk+/- by the trypan blue exclusion test. Afterwards, an in vitro mutagenicity and genotoxicity assessment of PTS has been performed using a prokaryotic system for the Ames test, and two different mammalian cell lines, L5178Y Tk+/- L5178Y Tk+/- (for MLA and MN tests) and Caco-2 cells (for the standard and enzyme-modified comet assay). 2. Materials and Methods 2.1. Supplies and chemicals Culture mediums (Minimum essential medium (MEM) and RPMI 1640 medium), cell culture reagents (, L-glutamine solution (CAS No. 56-85-9), sodium pyruvate solution (CAS No. 113-24-6), penicillin/streptomycin solution, gentamicin solution (CAS No 1405-41-0) and amphotericin B solution (CAS No. 1397-89-3) were from Gibco (Biomol, Sevilla, Spain)), and foetal bovine serum (FBS) and horse serum were obtained from Gibco (Biomol, Sevilla, Spain) BioWhittaker (Spain). PTS (95.5% purity) was kindly supplied by DOMCA S.L. (Granada, Spain). Field Code Changed Cyclophosphamide (CP, CAS No. 6055-19-2), mitomycin C (MMC, CAS No. 50-07-7), methyl methanesulfonate (MMS, 99% purity; CAS No. 66-27-3), hypoxanthine (99% purity; CAS No. 68- 94-0), thiazolyl blue tetrazolium bromide (MTT, 99.7% purity; CAS No. 298-93-1), MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium salt), and tetrazolium compound (CAS No. 351330-42-2) were purchased from (Promega (Biotech Ibérica,, Madrid, Spain); Coomassie Brilliant Blue G-250 (CAS No. 6104-59-2) was purchased from (BioRad, Madrid, Spain), trifluorothymidine (TFT, ≥ 99% purity; CAS No. 70-00-8), thymidine (CAS No. 4449-43-8), THMG medium (thymidine 9 µg/mL, methotrexate 0.3 µg/mL, hypoxantine 15 µg/mL, glycine 22.5 µg/mL) (CAS No. 59-05-2), glycine (≥99% purity; CAS No. 56-40-6), cythochalasin B (Cyt-B, 98%, CAS No. 14,930-96-2), Ggiemsa stain (CAS No. 51,811- 82-6), dimethyl sulfoxide (DMSO) (CAS No. 67-68-5), 2-nitrofluoerene (2-NF) (CAS No. 607-57- 8), sodium azide (NaN3) (CAS No. 26628-22-8), 2-aminofluorene (2-AF) (CAS No. 153-78-6), Neutral Red (CAS No. 553-24-2), and trypan blue solution 0.4% (CAS No. 72-57-1) were purchased from Sigma–Aldrich (Madrid, Spain). RPMI 1640 medium, horse serum, L-glutamine solution (CAS No. 56-85-9), sodium pyruvate solution (CAS No. 113-24-6), penicillin/streptomycin solution and amphotericin B solution (CAS No. 1397-89-3) were from Gibco (Biomol, Sevilla, Spain). S9 fraction was purchased from Moltox (Trinova, Biochem, Germany). Endo III (EC 3.1.21.5) was purchased from C-viral S.L. (Sevilla, Spain), and FPG (EC 3.2.2.23) from Sigma-Aldrich (Madrid, Spain). 2.2. Cells and culture conditions Caco-2 cells, used for the cytotoxicity and for the standard and enzyme-modified comet assays, are derived from a human colon carcinoma (ATCC© HTB-37). They were maintained at 37 °C in a humidified incubator gassed with 5% CO2 in air at 95% relative humidity (CO2 incubator, NuAire, Spain), in MEM supplemented with 1% non-essential amino acids (NEAA), 2 mM l-glutamine, 1.25 μg/mL fungizone, 1 mM pyruvate, 50 μg/mL gentamicin and 10% FBS. Cell viability and cell number were determined with the trypan blue exclusion test. Five S. typhimurium histidine-auxotrophic strains (TA97A, TA98, TA100, TA102 and TA104) were used for the Ames test, according to Organisation for Economic Cooperation and Development recommendations (OECD 471, 1997). For the MN and MLA tests, L5178Y Tk+/− mouse lymphoma cells were used. This cell line was originally provided by Dr. Olivier Gillardeux (Safoni-Synthélabo, Paris, France). L5178Y Tk+/- cells were cultured according to Mellado-Garcia et al. (2015). Cultures were maintained in a humidified incubator with 5% CO2 at 37 °C. To perform the assays, 2 × 105 cells/mL for the MN test and 1 × 107 cells/mL for the MLA were seeded. 2.3 Test solutions The concentration ranges of PTS were selected according the results obtained in the cytotoxicity assays carried out in Caco-2 and L5178Y Tk+/− cells. Stock solution of PTS (400 mM) was prepared in DMSO, and the different exposure concentration solutions were made by dilution in MilliQ sterile water (Ames test), RPMI 1640 medium (MN and MLA assays) or MEM medium (cytotoxicity, standard and modified comet assays). To avoid the toxic effects for the cells, the final percentages of DMSO were always less than 0.1% in all the assays. 2.4. Cytotoxicity assays. PTS cytotoxicity was measured in Caco-2 (to establish the EC50 for the comet assay) and L5178Y Tk+/- (for the MN test) cells, exposed to different concentrations of PTS (range 0– 500 μM) at two different exposure times (24 and 48 h). Caco-2 cells were plated at density of 7.5 × 105 cells/mL in 96 well tissue-culture plates. The basal cytotoxicity endpoints were the total protein content (TP), neutral red uptake (NRU) and 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) inner salt (Puerto et al., 2009). Discusión general/General discussion 174 demostraron que la administración de aceite de ajo en ratas durante 3 meses, inhibía significativamente el estrés oxidativo inducido en el cerebro por el NaNO2. De forma similar, inyecciones de extractos de ajo revirtieron los daños oxidativos inducidos por lindano (un insecticida organoclorado) en los testículos y el cerebro en ratas (Hfaiedh y cols., 2011). Además, el daño oxidativo producido en el hígado de ratas expuestas previamente a dicromato de potasio se pudo prevenir alimentándolas previamente con polvo de ajo (Pedraza-Chaverri y cols., 2008). De forma global, tras analizar todos los resultados obtenidos en el caso de PROALLIUM AP®, podemos afirmar que las concentraciones ensayadas parecen seguras con vistas a su futura comercialización en la industria alimentaria como ingrediente en los envases activos, permitiendo así aumentar la vida útil de los alimentos. Los estudios realizados por otros autores revelan una gran disparidad de resultados obtenidos en diferentes AEs o en sus componentes mayoritarios, usando distintos ensayos de mutagenicidad y genotoxicidad e incluso en estudios subcrónicos in vivo, en los cuales en ocasiones los átomos de azufre de los componentes juegan un papel clave en la protección del ADN frente a componentes externos. La existencia de todos estos estudios con resultados contradictorios refuerza la necesidad de realizar estudios toxicológicos previos para asegurar la salud de los consumidores. Los resultados derivados de esta Tesis Doctoral así como la búsqueda bibliográfica previa y la discusión con otros autores permiten obtener una visión más global de los hipotéticos daños que podrían ocasionar estos compuestos sobre la salud humana con la finalidad de establecer concentraciones de uso seguras para su empleo en la industria alimentaria. VI. Conclusiones / Conclusions Conclusiones/Conclusions 177 De los resultados obtenidos durante el desarrollo de esta Tesis Doctoral se ha llegado a las siguientes conclusiones: PRIMERA: La evaluación de la mutagenicidad in vitro de PTSO (OCDE 471) indicó una ausencia de mutagenicidad en las cepas de S. typhimurium tratadas; sin embargo, se observó daño mutagénico tras exposición a la línea L5178Y TK+/-en el ensayo de MLA (OCDE 476) tras 24h de exposición. SEGUNDA: La evaluación de la genotoxicidad in vitro de PTSO mostró resultados negativos en el ensayo de MN en células L5178Y TK+/- (OCDE 487) en ausencia de la fracción microsómica S9; sin embargo, en presencia de S9 sí se observó un incremento en la frecuencia de MN, indicando el potencial genotóxico de sus metabolitos. PTSO no produjo daños directos ni indirectos (oxidativos) del ADN a ninguna concentración en las células Caco-2, en el ensayo cometa estandar y ensayo enzimáticamente modificado . TERCERA: El estudio de la mutagenicidad in vitro de PTS indicó la ausencia de mutagenicidad en las cepas de S. typhimurium estudiadas, así como en el ensayo de MLA, con y sin presencia de activación metabólica. CUARTA: In vitro, PTS mostró genotoxicidad mediante el ensayo de MN tanto en ausencia como en presencia de la fracción microsómica S9. PTS no produjo roturas simples o dobles en el ADN, ni daños oxidativos, a ninguna concentración ensayada en los ensayos cometas realizados. QUINTA: La evaluación de la genotoxicidad in vivo en ratas Wistar de PTSO mostró ausencia de genotoxicidad tanto en el ensayo de MN (OCDE 474) como en el ensayo cometa (OCDE 489) en las ratas tratadas. Además, el uso de enzimas de restricción no reveló daño oxidativo en el ADN. El PTSO no fue detectado en el plasma de las ratas expuestas por la técnica UHPLC-MS/MS Orbitrap. Sin embargo, se demostró la presencia Conclusiones/Conclusions 178 de PTSO en las ratas expuestas mediante la identificación de sus componentes de degradación en estómago e hígado de las mismas, por Pirólisis analítica acoplada a un detector GC/MS (Py-GC/MS). Por otro lado, el ensayo histopatológico no mostró ningún daño en los órganos estudiados a la dosis más alta ensayada de PTSO (55 mg/kg). SEXTA: El estudio de toxicidad subcrónica in vivo de 90 días del PROALLIUM AP® (OCDE 408) no mostró cambios en el pe so de las ratas expuestas, ni en el consumo de agua y alimentos, así como tampoco se observaron diferencias significativas en los parámetros bioquímicos, hematológicos o histopatológicos respecto de las ratas control. Se estableció el NOAEL del PROALLIUM AP ® en 400 mg/kg/día en las condiciones de estudio. SÉPTIMA: El mecanismo de acción tóxica de PTSO no parece estar relacionado con la inducción de estrés oxidativo al no observarse producción de EROs en las células de leucemia monocítica humana THP-1 a las concentraciones ensayadas. No obstante, PTSO sí es capaz de provocar muerte celular en dichas células a concentraciones más elevadas a la que llegaría al consumidor en el peor escenario posible. Por último, a partir de la concentración a la que se observa mortalidad también se aprecia un aumento de la actividad fagocitaria de las células THP-1. OCTAVA: Tras los resultados obtenidos en todos los ensayos realizados en esta tesis doctoral, podemos concluir que el PROALLIUM AP® parece ser seguro para los consumidores a las concentraciones previstas para su potencial uso como antimicrobiano en los envases activos. Conclusiones/Conclusions 179 The following conclusions were reached from the results obtained during the development of this Doctoral Thesis: FIRST: The evaluation of in vitro mutagenicity of PTSO (OCDE 471) indicated an absence of mutagenicity in treated S. typhimurium strains; nevertheless, mutagenic damage was observed in L5178Y TK+/- cell lines after exposure in the MLA test (OCDE 476) after 24 hours of exposure. SECOND: The evaluation of in vitro genotoxicity of PTSO demonstrated negative results in MN assay with L5178Y TK+/- cells (OCDE 487) in absence of microsomic fraction S9. However, in presence of S9 an increase of MN frequency was observed, indicating genotoxic potential of metabolites. PTSO did not produce direct nor indirect damage (oxidative) to DNA at any concentration in Caco-2 cells, in standard comet assay and modified enzymatic assay. THIRD: The study of in vitro PTS mutagenicity indicated absence of mutagenicity in S. typhimurium assayed strains, as well as in MLA assay, with or without presence of metabolic activation. FOURTH: The In vitro, PTS demonstrated its genotoxicity by the MN assay both in absence and in presence of microsomic fraction S9. PTS did not produce simple nor double break in DNA strands, nor oxidative damage, at any concentration assayed in comet assays performed. Conclusiones/Conclusions 180 FIFTH: The evaluation of in vivo genotoxicity in Wistar rats of PTSO demonstrated absence of genotoxicity both in MN assay (OCDE 474) and in comet assay (OCDE 489) in treated rats. In addition, the use of restriction enzymes did not reveal oxidative damage in DNA. PTSO was not detected in rat plasma after exposure with the UHPLC-MS/MS Orbitrap technique. Nevertheless, the presence of PTSO was proven in exposed rats by identification of its degradation components in stomach and liver of rats, by analytical pyrolysis linked to a GC/MS detector (Py-GC/MS). Moreover, the histopathological study did not demonstrate any damage in studied organs at the highest assayed PTSO dose (55 mg/kg). SIXTH: The study of in vivo subchronic toxicity at 90 days of PROALLIUM AP® (OCDE 408) did not demonstrate weight changes of exposed rats, nor in water and food consumption, as well as no significant changes in biochemical, hematological or histopathological parameters respect to control rats. NOAEL of PROALLIUM AP ® was established at 400 mg/kg/day at the conditions assayed. SEVENTH: The toxic mechanism of action of PTSO does not appear to be related to the induction of oxidative stress. The production of ROS in the human monocytic leukemia cells THP-1 is not observed at the concentrations tested. However, PTSO is able to cause cell death in such cells at concentrations higher to those that are expected to reach the consumer in the worst possible scenario. Finally, from the concentration at which mortality is observed, there is also an increase in the phagocytic activity of THP-1 cells. Derived from the results obtained from all performed assays in this thesis, it can be concluded that PROALLIUM AP® seems to be safe for consumers under expected concentration for potential use as antimicrobial agent in active packaging. VIII. Referencias / Referencias Referencias/References 183 Abid-Essefi, S., Zaied, C., Bouaziz, C., Salem, IB., Kaderi, R., Bacha, H. 2012. Exp Toxicol Pathol. 64, 689-95. Protective effect of aqueous extract of Allium sativum against zearalenone toxicity mediated by oxidative stress. Amagase, H., Petesch, B.L., Matsuura, H., Kasuga, S., Itakura, Y., 2001. Intake of garlic and its bioactive components. The Journal of Nutrition, 131, 955S–962S. Arana-Sánchez, A., Estarrón-Espinosa, M., Obledo-Vázquez, EN., Padilla-Camberos, E., Silva- Vázquez, R., Lugo-Cervantes, E. 2010. Antimicrobial and antioxidant activities of Mexican oregano essential oils (Lippia graveolens H. B. K.) with different composition when microencapsulated in beta-cyclodextrin. Lett Appl Microbiol. 50, 585-590. Appendini, P. Hotchkiss, JH. 2002. Review of antimicrobial food packaging. Innovative Food Science and Emerging Technologies, 3, 113-126. Arranz, N., Haza, A.I., García, A., Möller, L., Rafter, J., Morales, P., 2007a. Protective effects of organosulfur compounds towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. Food Chem. Toxicol. 45, 1662-1669. Arranz, N., Haza, A.I.,, García, A., Delgado, E., Rafter, J., Morales, P., 2007b. Effects of organosulfurs, isothiocyanates and vitamin C towards hydrogen peroxide-induced oxidative DNA damage (strand breaks and oxidized purines/pyrimidines) in human hepatoma cells. Chemico-Biological Interactions. 169, 63–71. Amagase, H. Petesch, B.L. Matsuura, H. Kasuga, S. Itakura, Y. 2001. Intake of garlic and its bioactive components. The Journal of Nutrition, 131, 955S–962S. Amorati, R., Foti, M.C,. Valgimigli, L., 2013. Antioxidant activity of essential oils. J. Agric. Food Chem, 61, 10835–10847. Augusti, K.T., y Mathew, P.T., 1974. Lipid lowering effect of allicin (diallyl disulfide oxide) on long-term feeding in normal rats. Experientia, 30, 468-470. Bagheri F, Gol A, Dabiri S, Javadi A., 2011. Preventive effect of garlic juice on renal reperfusion injury. Iran J Kidney Dis. 5, 194-200. Bahram, S., Rezaei, M., Soltani, M., Kamali, A., Ojagh, S.M., Abdollahi, M., 2012. Whey protein concentrate edible film activated with cinnamon essential oil. J. Food Process. Pres, 38, 1251–1258. Referencias/References 190 Galotto,M. J., Valenzuela, X., Rodriguez, F., Bruna, J., & Guarda, A. 2012. Evaluation of the effectiveness of a new antimicrobial active packaging for fresh atlantic salmon (Salmo salar L.) shelf life. Packaging Technology and Science, 25, 363-372. Guyonnet, D., , C., Suschetet, M., Siess, M.H., Le bon, A.M., 2000. Liver subcellular fractions from rats treated by organosulfur compounds from Allium modulate mutagen activation. Mutat. Res., 466, 17–26. Guyonnet, D., Belloir, C., Suschetet, M., Siess, M.H., Le bon, A.M., 2001. Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction. Mutat. Res., 495, 135–145. Guarda, A., Rubilar, J.F., Miltz, J., Galotto, M.J., 2011. The antimicrobial activity of microencapsulated thymol and carvacrol. Int. J. Food Microbiol, 146, 144–150. Gutiérrez, L., Sánchez, C., Batlle, R., López, P. y Nerín, C. 2009. New Antimicrobial Active Package for Bakery Products. Trends in Food Science and Technology, 20, 92-99. Gutiérrez, L., Escudero, A., Batlle, R. y Nerín, C. 2010. Effect of Mixed Antimicrobial Agents and Flavors in Active. Packaging Films. Journal of Agricultural and Food Chemistry, 57, 8564- 8571. Hassanien, M.F., Assiri, A.M., Alzohairy, A.M,. Oraby, H.F. 2015. Health-promoting value and food applications of black cumin essential oil: an overview. J. Food. Sci. Technol., 52, 6136–6142. Han, J.H. y Floros, J.D. Casting antimicrobial packaging films and measuring their physical properties and antimicrobial activity, 1997. Journal. Plastic Film and Sheeting, 13, 287-298. Henriette M.C. 2009. Nanocomposites for food packaging application. Food Research International 42, 1240–1253. Hong, S.I, Park, J.D. y Kim, D.M. 2000. Antimicrobial and physical properties of food packaging films incorporated with some natural compounds. Food Science Biotechnology 9, 38-42. Hotchkiss, J. 1997 . Food packaging interactions influencing quality and safety.Food Addities and Contaminants, 1467 , 601-607. Hu, C.Y., Chen, M., Wang, Z.W., 2012. Release of thymol, cinnamaldehyde and vanillin from soy protein isolate films into olive oil. Packag. Technol. Sci, 25, 97–106. Hyldgaard, M., Mygind, T., Meyer, R.L., 2012. Essential oils in food preservation: mode of action, synergies, and interaction with food matrix components. Front. Microbiol, 3, 1- 24. Referencias/References 191 ICH harmonised tripartite guideline. guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use s2(r1). Current Step 4 version dated 9 November 2011. Informe sobre envases activos e inteligentes, Pacman 2013. Promoción de la Competitividad, la Atracción y la Internacionalización en los Clusters Agroalimentarios del Área Mediterránea. Pp: 1-111. Informe de vigilancia tecnológica, tendencias en la tecnología del envasado activo, 2015. Escuela de vigilancia industrial, Fundación EOI. Ministerio de Industria, Energía y Turismo, pp 1-110. Ishikawa, K., Naganawa, R., Yoshida, H., Iwata, N., Fukuda, H., Fujino, T., Suzuki, A., 1996. Effects of Ajoene,an organosulfur compound derived from garlic. Biosci.Biotech.Biochem., 60, 2086-2088. Jeena, K., Liju, VB., Kuttan, R., 2011. A preliminary 13-week oral toxicity study of ginger oil in male and female Wistar rats. Int J Toxicol. 30, 662-670. Jouki, M., Yazdi, F. T., Mortazavi, S. A., Koocheki, A., & Khazaei, N. 2014. Effect of quince seed mucilage edible films incorporated with oregano or thyme essential oil on shelf life extension of refrigerated rainbow trout fillets. International Journal of Food Microbiology, 174, 88-97. Kada, T., Sadaie ,Y., Hara, M., 1978. Analysis of mutagen-antimutagen reactions in food and food additives by the rec-assay and reversion-assay procedures. Mutation Research 53, 206-207. Kang, SH., Kwon, JY., Lee, JK., Seo, YR., 2013. Recent advances in in vivo genotoxicity testing: prediction of carcinogenic potential using comet and micronucleus assay in animal models. J Cancer Prev. 18, 277-88. Kerry, JP. O’Grady, MN. Hogan, SA, 2006. Past, current and potential utilisation of active and intelligentpackaging systems for meat and muscle-based products: A review. Meat Science 74, 113–130 Kirkland D, Reeve L, Gatehouse D, Vanparys P, 2011. A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins. Mutat. Res. 721, 27-73. Kirkland, D., Zeiger, E., Madia, F., Corvi, R.,2014. Can in vitro mammalian cell genotoxicity test results be used to complement positive results in the Ames test and help predict carcinogenic or in vivo genotoxic activity? II. Construction and analysis of a Referencias/References 192 consolidated database. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 775-776, 69-80. Kohda, K., Goda, H., Itoh, K., Samejima, K., Fukuuchi, T. 2013. Aged Garlic Extract Reduces ROS Production and Cell Death Induced by 6-Hydroxydopamine through Activation of the Nrf2-ARE Pathway in SH-SY5Y Cells Pharmacology & Pharmacy, 4, 31-40. Kirkland, D., Pfuhler, S., Tweats, D., Aardema, M., Corvi, R., Darroudi, F., Elhajouji, A., Glatt, H., Hastwell, P., Hayashi, M., Kasper, P., Kirchner, S., Lynch, A., Marzin, D., Maurici, D., Meunier, JR., Müller, L., Nohynek, G., Parry, J., Parry, E., Thybaud, V., Tice, R., van Benthem, J., Vanparys, P., White, P. 2007. How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: report of an ECVAM Workshop. Mutat. Res., 628, 31–55. Kirkland, D., Reeve, L., Gatehouse, D., Vanparys, P., 2011. A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins. Mutat. Res. 721, 27-73. Knasmüiller, S., de Martin R., Domjan, G., Szakmary, A., 1989. Studies on the antimutagenic activities of garlic extract. Environmental and Molecular Mutagenesis 13, 357 365. Kuorwel, K., Cran, M.J., Sonneveld, K., Miltz, J., Bigger, S.W., 2011. Antimicrobial activity of biodegradable polysaccharide and protein-based films containing active agents. J. Food Sci, 76, 90–102. Lanzotti, V., 2006. The analysis of onion and garlic. J. Chromatogr. A. 1112, 3-22. Lasrado, J.A., Trinker, D., Ceddia, M.A., Herrlinger, K.A., 2015. The safety of a dry spearmint extract in vitro and in vivo. Regul Toxicol. Pharm., 71, 213–224. Liju, V.B., Jeena, K., Kuttan, R., 2013. Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric (Curcuma longa L.), Food Chem. Toxicol. 53, 52– 61. López-Rubio, A. Almenar, E. Hernandez-Muñoz, P. Lagarón, JM. Catalá, R. Gavara, R., 2004. Overview of Active Polymer-Based PackagingTechnologies for Food Applications. Food Reviews International. 20, 357–387. López-Rubio, A. Gavara, R. Lagaron, JM. 2006. Bioactive packaging: turning foods into healthier foods through biomaterials. Trends in Food Science and Technology, 17, 567-575. Lancaster, J.E., y Shaw, M.L. 1989. G-Glutayl peptides in the biosynthesis of S-alk(en)yl-L- cysteine sulfoxides (flavor precursors) in Allium. Phytochemistry, 28, 455-460. Referencias/References 193 Llana-Ruiz-Cabello, M., Maisanaba, S., Gutiérrez-Praena, D., Prieto, AI., Pichardo, S., Jos, A., Moreno, F.J., Cameán, AM. 2015a. Cytotoxic and mutagenic in vitro assessment of two organosulfur compounds derived from onion to be used in the food industry. Food Chemistry. Volume 166, 1 Pages 423–431. Llana-Ruiz-Cabello, M. Gutiérrez-Praena, D. Puerto, M. Pichardo, S. Moreno, F.J. Baños, A. Nuñez, C. Guillamón, E. Cameán, A.M. 2015b, Acute toxicological studies of the main organosulfur compound derived from Allium sp. intended to be used in active food packaging. Food Chem. Toxicol., 82, pp. 1–11. Llana-Ruiz-Cabello, M., Pichardo, S., Maisanaba, S., Puerto, M., Prieto, A.I., Gutiérrez-Praena, D., Jos, A., Cameán, A.M., 2015c. In vitro toxicological evaluation of essential oils and their main compounds used in active food packaging: A review. Food and Chemical Toxicology 81, 9–27. Liao, W., McNutt, M.A., Zhu, Wei-Guo., 2009. The comet assay: A sensitive method for detecting DNA damage in individual cells. Methods Volume 48, 46–53. López, P., Sánchez, C., Batlle, R., Nerín, C., 2007. Development of flexible antimicrobial films using essential oils as active agents. J. Agric. Food Chem, 55, 8814–8824. Maisanaba, S., Llana-Ruiz-Cabello, M., Gutiérrez-Praena, D., Pichardo, S., Puerto, M., Prieto, A.I. Jos A. Cameán, A.M. 2016. New advances in active packaging incorporated with essential oils or their main components for food preservation. Food Reviews International, 1-69. Marcos, B., Aymerich, T., Garriga, M., Arnau, J. 2013. Active packaging containing nisin and high pressure processing as post-processing listericidal treatments for convenience fermented sausages. Food Control, 30, 325-330. Martínez-Fernández, G., Abecia, L., Martín-García, AI., Ramos-Morales, E., Hervás, G., Molina- Alcaide, E., Yáñez-Ruiz, DR., 2013. In vitro-in vivo study on the effects of plant compounds on rumen fermentation, microbial abundances and methane emissions in goats. Animal 7, 1925-34. Martínez-Fernández, G., Abecia, L., Martín-García, AI., Ramos-Morales, E., Denman, SE., Newbold, CJ., Molina-Alcaide, E., Yáñez-Ruiz, DR., 2015. Response of the rumen archaeal and bacterial populations to anti-methanogenic organosulphur compounds in continuous-culture fermenters. FEMS Microbiol. Ecol. 91, 79. Referencias/References 194 Manso, S., Pezo, D., Gómez-Lus, R., Nerín, C. 2014. Diminution of aflatoxin B1 production caused by an active packaging containing cinnamon essential oil. Food Control, 45, 101– 108. Marks, HS., Anderson, JL., Stoewsand, GS., 1992. Inhibition of benzo[a]pyrene-induced bone marrow micronuclei formation by diallyl thioethers in mice. J Toxicol Environ Health. 37, 1-9. Marks, H.S., Anderson, J.A. Stoewsand G.S. 1993. Effect of s-methyl cysteine sulphoxide and its metabolite methyl methane thiosulphinate, both occurring naturally in brassica vegetables, on mouse genotoxicity. Food and Chemical Toxicology. 7, 491-495. Marsh, K. y Bugusu, B. Food Packaging— Journal of food science 2007. Vol. 72, Nr. 3, 39-54 Mezzoug, N., Elhadri, A., Dallouh, A., Amkiss, S., Skali, N.S., Abrini, J., Zhiri, A., Baudoux, D., Diallo, B., El Jaziri, M., Idaomar M., 2007. Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents. Mutat. Res, 629, 100–110. Milner, J A. A historical perspective on garlic and cáncer. The Journal of Nutrition, supl. Recent Advances on the Nutritional Effects Associated, 131,3S 1027S-1031S. Miller, HE., Rigelhof, F., Marquart, L., Prakash, A., Kanter, M.2000. Antioxidant content of whole grain breakfast cereals, fruits and vegetables. J Am Coll Nutr. 19, 1–8. Millette, M., Le Tien, C., Smoragiewicz, W., y Lacroix, M. 2007. Inhibition of Staphylococcus aureus on beef by nisin-containing modified alginate films and beads. Food Control, 18, 878-884. Mortelmans, K., Zeiger, E., 2000. The Ames Salmonella/microsome mutagenicity assay. Mutat. Res. 445, 29–60. Musah, R., Lesiak, AH., Maron, MJ., Robert, B., Cody, DE., Kristen, L., Fowble, A., Dane, J., Long, MC., 2016. Mechano sensitivity below Ground: Touch-Sensitive Smell-Producing Roots in the Shy Plant Mimosa pudica. American Society of Plant Biologist. 170, 1075–1089. Musk, S.R.R., Clapham, P., Johnson, I.T., 1997. Cytotoxicity and genotoxicity of diallyl sulfide and diallyl disulfide towards Chinese hamster ovary cells. Food Chem. Toxicol., 35, 379–385. Referencias/References 195 Nakagawa, H.T., Tsuta, K., Kiuchi, K., Senzaki, H., Tanaka, K., Hioki, K., Tsubura, A., 2001. Growth inhibitory effects of diallyl disulfide on human breast cancer cell lines. Carcinogenesis 22: 891–897. Natrajan, N. y Sheldon, B.W. 2000. Efficacy of NisinCoated Polymer Films To Inactivate Salmonella Typhimurium on Fresh Broiler Skin. Journal of Food Protection, 63, 1189-1196. Nuutila, AM., Puupponen-Pimia, R., Aarni, M., Oksman-Caldentey, KM., 2003. Comparison of antioxidant activities of onion and garlic extracts by inhibition of lipid peroxidation and radical scavenging activity. Food Chem 81,485–93. De Martino, A., Filomeni, G., Aquilano, K., Ciriolo, M.R., Rotilio, G., 2006. Effects of water garlic extracts on cell cycle and viability of HepG2 hepatoma cells. J. Nutr. Biochem. 17, 742– 749. Nerín, C. Tovar, L. y Salafranca, J. 2008. Behaviour of a new antioxidant active film versus oxidizable model compounds. Journal of Food Engineering, 84, 313-320. Nerín, C. 2010. Antioxidant active packaging and antioxidant edible films, “Oxidation in foods and beverages and antioxidant applications”, E Decker, R Elias (eds.), Woodhead Publishing Cambridge, United Kingdom, 496-515. Nguyen, VT., Gidley, M. J., Dykes, GA. 2008. Potential of a nisin-containing bacterial cellulose film to inhibit Listeria monocytogenes on processed meats. Food Microbiology, 25, 471-478. OECD (Organisation for Economic Cooperation and Development) Guideline 407, Guideline for Testing of Chemicals: Repeated Dose 28-Day Oral Toxicity Study in Rodents, 2008, pp. 1-13. OECD (Organisation for Economic Cooperation and Development) Guideline 408, Guideline for Testing of Chemicals: Repeated Dose 90-day Oral Toxicity Study in Rodents 1998, pp. 1- 10. OECD (Organisation for Economic Cooperation and Development) Guideline 414, Guideline for Testing of Chemicals: Prenatal Developmental Toxicity Study, 2011, pp. 1-11. OECD (Organisation for Economic Cooperation and Development) Guideline 416, Guideline for Testing of Chemicals: Two-Generation Reproduction Toxicity Study, 2001, pp. 1-13. OECD (Organisation for Economic Cooperation and Development) Guideline 417, Guideline for Testing of Chemicals: Toxicokinetics, pp. 1.20. OECD (Organisation for Economic Cooperation and Development) Guideline 424, Guideline for Testing of Chemicals: Neurotoxicity Study in Rodents, 1999, pp. 1-15. Referencias/References 196 OECD (Organisation for Economic Cooperation and Development) Guideline 426, Guideline for Testing of Chemicals: Developmental Neurotoxicity Study, 2007, pp. 1-26. OECD (Organisation for Economic Cooperation and Development) Guideline 443, Guideline for Testing of Chemicals: Extended One-Generation Reproductive Toxicity Study, 2011, pp. 1-25. OECD (Organisation for Economic Cooperation and Development) Guideline 451, Guideline for Testing of Chemicals Carcinogenicity Studies, 2009, pp. 1-1 OECD (Organisation for Economic Cooperation and Development) Guideline 452, Guideline for Testing of Chemicals: Chronic Toxicity Studies, 2009, pp. 1-16. OECD (Organisation for Economic Cooperation and Development) Guideline 453, Guideline for Testing of Chemicals: Combined Chronic Toxicity\Carcinogenicity Studies, 2009, pp. 1-20. OECD (Organisation for Economic Cooperation and Development) Guideline 471, Guideline for Testing of Chemicals: Mutation Test 1997, pp. 1–11. OECD (Organisation for Economic Cooperation and Development) Guideline 474, Guideline for the testing of chemicals: mammalian erythrocyte micronucleus test 2014, pp. 1–21. OECD (Organisation for Economic Cooperation and Development) Guideline 486, Guideline for the testing of chemicals: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo 1997, pp. 1-8. OECD (Organisation for Economic Cooperation and Development) Guideline 487, Guideline for Testing of Chemicals: in vitro Mammalian Cells Micronucleus Test 2014, pp. 1–26. OECD (Organisation for Economic Cooperation and Development) Guideline 488, Guideline for Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays, 2011, pp.1-15. OECD (Organisation for Economic Cooperation and Development) Guideline 489, Guideline for the testing of chemicals: in vivo mammalian alkaline comet assay 2014, pp. 1–25. Orban, J.I., Patterson, J.A., Sutton, A.L., y Richards, G.N., 1997. Effect of sucrose thermal oligosaccharide caramel, dietary vitamin-mineral level, and brooding temperature on growth and intestinal bacterial populations of broiler chickens. Poult. Sci. 76:482–490. Referencias/References 197 Ou, CC., Tsao, SM., Lin, MC., Yin, MC. 2003. Protective action on human LDL against oxidation and glycation by four organosulfur compounds derived from garlic. Lipids 38, 219–24. Ouattara, B., Simard, R.E., Piette, G., Begin, A. y Holley, R.A. 2000. Diffusion of acetic and propionic acids from chitosan-based antimicrobial packaging films. Journal of Food Science, 65, 768-773. Ozdemir, M. y Floros, JD. Active Food Packaging Technologies. Critical Reviews in Food Science and Nutrition, 44, 185–193. Pedraza-Chaverrí, J., González-Orozco, AE., Maldonado, PD., Barrera, D., Medina-Campos, ON., Hernández-Pando, R., 2003. Diallyl disulfide ameliorates gentamicin-induced oxidative stress and nephropathy in rats. European Journal of Pharmacology, 473, 71–78. Peinado, M.J. Ruiz, R. Echávarri, A. Rubio, L.A. 2012. Garlic derivative propyl propane thiosulfonate is effective against broiler enteropathogens in vivo. Poult. Sci., 91, 2148– 2157. Peinado, M.J., Ruiz, R., Echávarri, A., Aranda-Olmedo, I., Rubio, L.A., 2013. Garlic derivative PTS-O modulates intestinal microbiota composition and improves digestibility in growing broiler chickens. Anim. Feed Sci. Technol., 181, 87–92. Pezo, C., Tovar, L. y Salafranca, J. 2008. Determination of the antioxidant capacity of active food packagings by in situ gas-phase hydroxyl radical generation and high-performance liquid chromatography–fluorescence detection. Journal of Chromatography A, 1178, 126-133. Ray, B., Chauhan, NB., Lahiri, DK. 2011. Oxidative insults to neurons and synapse are prevented by aged garlic extract and S–allyl–L–cysteine treatment in the neuronal culture and APP– Tg mouse model. J Neurochem. 117, 388-402. REGLAMENTO (CE) No 1935/2004 DEL PARLAMENTO EUROPEO Y DEL CONSEJO, de 27 de octubre de 2004, sobre los materiales y objetos destinados a entrar en contacto con alimentos. www.boe.es/doue/2004/338/L00004-00017.pdf Restucciaa, D. Gianfranco, U. Spizzirria, Parisi, O. Cirillo, G. Curcio. M, Iemma, F. Puoci, F. Vinci, G. Picci, N., 2010. New EU regulation aspects and global market of active and intelligent packagingfor food industry applications. Food Control 21, 1425–1435. Referencias/References 198 Realini, C., Marcos, R. Active and intelligent packaging systems for a modern society. (2014). Meat Science 98, 404–419. Ribnicky, D.M., Poulev, A., O'Neal, J., Wnorowski, G., Malek, D.E., Jäger, RRaskin. I., 2004. Toxicological evaluation of the ethanolic extract of Artemisia dracunculus L. for use as a dietary supplement and in functional foods. Food Chem. Toxicol., 42, 585–598. Roby, M.H.H. Sarhana, M.A. Selima, K.A.H. Khalela, K.I. 2013. Evaluation of antioxidant activity, total phenols and phenolic compounds in thyme (Thymus vulgaris L.), sage (Salvia officinalis L.), and marjoram (Origanum majorana L.) extracts. Ind. Crops Prod, 43, 827– 831. Rodriguez, A., Batlle, R. y Nerin, C. 2007. The use of natural essential oils as antimicrobial solutions in paper packaging. Progress in Organic Coatimgs, 60, 33-38. Rodríguez, A., R. Batlle. y C. Nerín 2008. The use of cinnamon-based active paper packaging against Rhizopus stolonifer food spoilage. Journal of Agricultural and Food Chemistry, 56, 6364-6369. Rodríguez-Lafuente, A., Nerin, C., Batlle, R., 2010. Active paraffin-based paper packaging for extending the shelf life of cherry tomatoes. J. Agric. Food Chem, 58, 6780–6786. Romeilah, R.M. Fayed, S.A. Mahmoud, G.I. 2010. Chemical compositions, antiviral and antioxidant activities of seven essential oils. Journal of Applied Sciences Research, 6, 50– 62. Rooney, ML. Introduction to active packaging technologies. In: Han JH, editor, 2005. Innovation in Food Packaging. Elsevier Academic Press; p, 63. Rose, P., Matt, W., Philip, K., Mooreband, Y., Zhun, Zh., 2005. BioactiveS-alk(en)yl cysteine sulfoxide metabolites in the genus Allium: the chemistry of potential therapeutic agents. Nat. Prod. Rep., 22, 351–368. Ruiz, R., Peinado, M. J., Aranda-Olmedo, I., Abecia, L., Suárez-Pereira, E., Ortiz Mellet, C., García Fernández, J.M., Rubio, L. A., 2015. Effects of feed additives on ileal mucosa– associated microbiota composition of broiler chickens. J. Anim. Sci. 93, 3410-3420. Sacchetti, G., Maietti, S., Muzzoli, M., Scaglianti, M., Manfredini, S., Radice, M., Bruni, R. 2005. Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chem, 91, 621–632. Referencias/References 199 Sanla-Ead, N., Jangchud, A., Chonhenchob, V., Suppakul, P., 2012. Antimicrobial activity of cinnamaldehyde and eugenol and their activity after incorporation into cellulose-based packaging films. Packag. Technol. Sci, 25, 7–17. Santoro, GF., das Graças, M., Guimarães, LG., Salgado, AP., Menna-Barreto, RF., Soares, MJ. 2007. Effect of oregano (Origanum vulgare L.) and thyme (Thymus vulgaris L.) essential oils on Trypanosoma cruzi (Protozoa: Kinetoplastida) growth and ultrastructure. Parasitol Res. 100, 783-90. Sapunjieva, T., Alexieva, I., Mihaylova, D., Popova, A.,2012. Antimicrobial and antioxidant activity of extracts of Allium ursinum L. J BioSci Biotech, 143-145. Saverini, M., Catanzaro, I., Sciandrello, G., Avellone, G., Indelicato, S., Marcì, G., Palmisano, L., 2012. Genotoxicity of citrus wastewater in prokaryotic and eukaryotic cells and efficiency of heterogeneous photocatalysis by TiO(2). J Photochem Photobiol B. 108, 8- 15. Scharff, R.L. 2012. Economic burden from health losses due to foodborne illness in the United States. Journal of Food Protection, 75, 123–131. Seow, Y.X., Yeo, C.R., Chung, H.L., Yuk, H.G., 2014. Plant essential oils as active antimicrobial agents. Crit. Rev. Food Sci. Nutr, 54, 625–644. Shin, H.A. Cha, Y.Y. Park, M.S. Kim, J.M. Lim, Y.C. 2010. Diallyl sulfide induces growth inhibition and apoptosis of anaplastic thyroid cancer cells by mitochondrial signaling pathway. Oral Oncology, 46, e15–e18. Shon, M.Y., Choi, S.D., Kahng, G.G., Nam, S.H., Sung, N.J., 2004. Antimutagenic, antioxidant and free radical scavenging activity of ethylacetate extracts from white, yellow and red onions. Food Chem. Toxicol, 42, 659–666. Shukla, Y., Taneja, P., 2012. Antimutagenic effects of garlic extract on chromosomal aberrations. Cancer Letters 176, 31–36. Sinha, S., Jothiramajayam, M., Ghosh, M., Mukherjee, A., 2014. Evaluation of toxicity of essential oils palmarosa, citronella, lemongrass and vetiver in human lymphocytes. Food and Chemical Toxicology 68, 71–77. Stajner, D., Popović, BM., Canadanović-Brunet, J., Stajner, M. 2008. Antioxidant and scavenger activities of Allium ursinum. Fitoterapia. 2008, 79: 303-305. Skandamis, P. N. y Nychass, G.J. 2002. Preservation of fresh meat with active and modified atmosphere packaging conditions. International Journal of Food Microbiology, 79, 35-45.