Safety assessment of propyl-propane-thiosulfonate (PTSO): 90-days oral subchronic toxicity study in rats

Sa e y assessmen o p opyl-p opane- hiosul ona e (PTSO): 90-days o al subch onic oxici y s udy in a s Au o es: An onio Cascajosa Li a1, Ana Isabel P ie o1, Albe o Baños2, En ique Guillamón2, Rosa io Moyano3 , Angeles Jos1*, Ana M. Cameán1. 1A ea o Toxicology, Facul y o Pha macy, Uni e si y o Se illa, Se illa, Spain. 2 DMC Resea ch Cen e SLU, Camino de Jayena s/n, Alhedin, 18620 G anada, Spain. 3 Depa men o Pha macology, Toxicology and Legal and Fo ensic Medicine, Uni e si y o Co doba, Campus de Rabanales Ca e e a Mad id-Cadiz s/n *Co esponding au ho : Ángeles Jos Gallego A ea o Toxicology, Facul y o Pha macy, Uni e sidad de Se illa. C/ P o eso Ga cía González, nº 2. C.P. 41012, Se ille. Spain. E-mail add ess: [email p o ec ed] Manusc ip Ti le Page - A 90 days o al oxici y s udy was conduc ed wi h p opyl-p opane- hiosul ona e - PTSO did no induce oxic e ec s a he highes dose assayed (55 mg/Kg/day) - NOAEL o PTSO was es ima ed o be ≥ 55 mg/Kg/day Highligh s ( o e iew) Abs ac P opyl-p opane- hiosul ona e (PTSO) is one o he main o ganosul u compounds p esen in Allium essen ials oil. Di e en applica ions in he ood sec o ha e been p oposed o PTSO, such as ood and eed addi i e and as ac i e packaging. Howe e , he au ho iza ion o i s use depends on i s oxici y p o ile. Thus, as a pa o i s sa e y assessmen , in his wo k a epea ed dose 90-day o al oxici y s udy has been conduc ed o he i s ime in a s ollowing he OECD guideline 408. PTSO was adminis e ed o g oups o 10 male and 10 emale a s a dose le els o 0, 14, 28, and 55 mg/Kg/day. No clinical signs o mo ali y and no changes in body weigh , ood consump ion and eed con e sion e iciency we e de ec ed h ough he s udy. Mo eo e , no ea men - ela ed changes in hema ological and biochemical pa ame e s we e obse ed, o ei he sex o dose g oups. The his opa hology s udy pe o med e ealed no di e ences in o gan weigh s, and no mo phological and his opa hological changes we e obse ed. Based on hese esul s, he no-obse ed-ad e se-e ec le el (NOAEL) o PTSO was judged o be ≥ 55 mg/Kg/day o bo h sexes. Keywo ds: Subch onic oxici y; 90-day; O ganosul u compounds; Allium; P opyl- p opane hiosul ona e Abs ac 1 1. In oduc ion Essen ials Oils (EO) ex ac s om plan s ha e been he ocus o nume ous s udies due o hei po en ial in he pha maceu ical and ood indus ies. Se e al wo ks and pa en s ha e been de eloped speci ically o he applica ion o EO and hei componen s in o he ood sec o (Ribei o-San os e al., 2017; Llana-Ruiz- Cabello e al., 2015; Maisanaba e al., 2017). Globally, hei ela i ely sa e s a us, p ope ies and accep ance by consume s which demand na u al compounds o eplace syn he ic ones ha e piqued he in e es o indus ies and consume s (Sacche i e al., 2005; Benkeblia and Lanzo i, 2007; Debiagi e al., 2014). Among hose bene icial plan s, Allium sp. is a genus well- known o i s an imic obial, an i i al, an ip o ozoal, an i ungal o an ioxidan p ope ies (among o he s). These p ope ies a e mainly due o hei con en o o ganosul u compounds (OSC), which a e seconda y phy ochemical me aboli es, biosyn hesized o de ensi e pu poses agains bio ic and abio ic s esso s. They a e mainly o med o he ac ion o he enzyme alliin (s o ed in acuoles) on cy oplasmic compounds like alk(en)yl cys eine sul oxides (ACOs) once he ege able issue is hu (Pu nik e al., 2019) (Fig. 1). O he wise, hese compounds a e no a ailable in in ac cells as hey a e oxic o he plan (Rami ez e al., 2017). One o he main componen s o Allium essen ial oil is p opyl-p opane- hiosul ona e (PTSO), ha co esponds o he molecula o mula C6H14O2S2 (Fig. 1). This compound has been s abilized and cha ac e ized by DMC Resea ch Cen e SLU ( G anada, Spain) o be used o di e en applica ions, aking ad an age o i s bene icial p ope ies. Thus, his p oduc has been epo ed o show mainly an ioxidan and an imic obial ac i i ies, being able o inhibi he g ow h o G am (–) and G am (+) bac e ia as well as molds and yeas (Peinado e al., 2012, 2013; Llana-Ruiz-Cabello e al., 2015). I s an ibac e ial ac i i y in i o in humans has also been demons a ed (So lozano-Pue o e al., 2018). Manusc ip o e ision (CLEAN VERSION) Click he e o iew linked Re e ences 2 P oallium AP®, a comme cial Allium sp. ex ac wi h a 14.5% PTSO con en , has been p oposed as a biop ese a i e in ac i e ood packaging o human ood commodi ies mainly due o i s an ioxidan and an imic obial ac i i y (Llana-Ruiz- Cabello e al., 2018). The packaging ma e ial used in hese sys ems can inco po a e componen s in ended o be eleased in o he ood om he package, allowing oods o a i e a he consume s wi h hei o iginal o enhanced o ganolep ic p ope ies, wi h longe shel -li e and sa e y (Ribei o-San os e al., 2017). P e iously, ano he s udy ca ied ou by Seydim & Sa ikus, (2006) es ed he an imic obial ac i i y o ga lic EO in combina ion wi h o egano EO in ilms made wi h whey p o ein isola e, and showed an imic obial ac i i y in a concen a ion o 4% (w/ ). Bu he inco po a ion o ga lic EO and hei componen s in ac i e ood packaging can esul in a highe human exposu e and consequen ly, mo e esea ch is needed o s ablish he sa e y concen a ion. An addi ional p oposed applica ion o PTSO is as senso y addi i e in animal nu i ion, imp o ing he pala abili y o eed, and also as a zoo echnical addi i e, being an al e na i e o he use o an ibio ics (Peinado e al., 2012), con ibu ing o educe esis ance gene a ed by hei excessi e use in li es ock. Since he ban in he Eu opean Union (EU) o he use o an ibio ics as g ow h p omo e s, he sea ch o new al e na i e p oduc s ha ensu e simila p oduc ion le els and ood secu i y wi hou gene a ing unwan ed e ec s, including human esis ances, has been os e ed, being addi i es o na u al o igin a good al e na i e. The egula ion (EC) No 1831/2003 o he Eu opean Pa liamen and o he Council o 22 Sep embe 2003 se s he pa ame e s o use o he addi i es used in animal nu i ion. Recen s udies ha e shown ha some eed addi i es can help animals o main ain good physiological condi ions and imp o e animal wel a e. This has led o an amendmen in he abo emen ioned egula ion on 12 h June 2019, es ablishing new unc ional g oups o eed addi i es o imp o e 3 physiological condi ion o animals. In his ega d, o he p ope ies demons a ing he po en ial use o PTSO in animal nu i ion sec o a e he an i-me hanogenic e ec desc ibed du ing he e men a ion p ocess in umen (Ma ínez-Fe nández e al. 2013 ) and he immunomodula o y and an i-in lamma o y p ope ies (Vezza e al., 2019). Howe e , he success ul de elopmen o any applica ion o his compound mus be accompanied by an ex ensi e oxicological e alua ion, ha gua an ees i s sa e y o he inal consume s, bo h humans and li es ock. In his ega d, he oxicological p o ile o PTSO has been in es iga ed by cy o oxici y assays in human cell lines (Llana-Ruiz-Cabello e al., 2015) as well as i s geno oxici y and mu agenici y in i o (Mellado-Ga cía e al., 2015) and geno oxici y in i o (Mellado-Ga cia e al., 2016b). Fu he mo e, i s acu e oxici y in i o has been also e alua ed (Llana-Ruiz-Cabello e al., 2015). No wi hs anding, sa e doses o use ha e been no ye es ablished o PTSO. Thus, a epea ed dose 90-days o al oxici y s udy in oden s o PTSO would be necessa y o clea ly cha ac e ize i s oxici y, being also a equi emen in he au ho iza ion applica ion p ocesses o he Eu opean Food Sa e y Au ho i y (EFSA, 2012; 2016a; 2016b; 2017). The aim o he p esen s udy is, he e o e, o u he explo e he oxici y po en ial o PTSO and, o he i s ime, o conduc a subch onic die a y oxici y assay o PTSO in a s ollowing in e na ionally ecognized es guidelines (O ganisa ion o Economic Co-ope a ion and De elopmen , OECD 408, 2018). Conside ing hese ac s, in he p esen s udy se e al pa ame e s ha e been e alua ed, including body weigh changes, ood and wa e consump ion, eed con e sion e iciency, o gan weigh a ios and biochemis y and hema ology pa ame e s. In addi ion he his opa hology o a ious issues has been also s udied. The esul s om he comple e assessmen o his 4 subch onic s udy would allow o ge an es ima ion o a non-obse ed-ad e se-e ec - le el (NOAEL) o exposu e o es ablish sa e y condi ions o human exposu e o PTSO. 2. Ma e ials and Me hods 2.1. Tes i em and doses p epa a ion PTSO was supplied by DMC RC SLU (G anada, Spain) wi h a 96% o pu i y. Comme cial powde neu al gela in om po k p o ein (Jesus Na a o S.A., Alican e, Spain) was employed as he ehicle o he es subs ance in all g oups including he con ols. Fo he 90-days s udy, he doses we e p epa ed daily o each animal du ing he 13 weeks, and on F idays hey we e also p epa ed o he weekend. The dose was mixed in 3 mL o liquid gela in. The olumes o PTSO (µL) o add o he gela in depended on he dose selec ed o each g oup, and he gela in could solidi y a 4 ºC o e nigh . Homogenei y o he die a y dose o mula ions and hei s abili y we e con i med o be a leas 5 days. 2.2. Animals condi ions and husband y se ices The a s we e supplied by Cha les Ri e labo a o ies S.L. (Kings, NY, USA), 40 males and 40 emales o Sp ague-Dawley s ain. They we e app oxima ely 7 weeks old and we e s abilized o an acclima iza ion pe iod o 7 days du ing which hey we e examined by a e e ina y su geon. When he i s week o dosage began he a s body weigh mean was 320 g ± 11.3 o males and 227 ± 11.9 o emales. 5 Animals we e indi idually housed in cages ype 3H wi h Sou ali 29/12 plus (sou ali S.L., Ge ona, Spain) aspen wood bedding and ood comple ely a ailable wi hou es ic ion using s anda d d y pelle die o oden s Scien i ic (Panlab, S.L.U., Co nella de Llob ega , Ba celona, Spain). They we e kep in a oom wi h con olled condi ions o hygiene behind a ba ie sys em, a ange o empe a u e o 21±2ºC, wi h a 10-15 ai changes pe hou , and a ela i e humidi y be ween 30-70% unde 12 h ligh /da k cycle. Each cage con ained an in o ma ion ca d which con ained s udy code (19-CAM-11-animal numbe ), sex, dose, g oup, and indi idual animal iden i ica ion. Communi y ap wa e (EMACSA, Co doba Wa e company, Có doba, Spain), il e ed and au ocla ed was a ailable ad libi um. 2.3. S udy design The maximum ole able dose (MTD) o PTSO in a s o ally exposed o PTSO (ga age) was p e iously se a 55 mg/kg by Llana-Ruiz-Cabello e al., (2015) ollowing he OECD 425 (2008) es guideline (o al oxici y s udy: Up and Down p ocedu e), and i was used as a e e ence o es ablish he es doses. Acco dingly, his dose was selec ed as he highes one o be es ed, and also descending doses using a 2- old in e al ac o acco ding o he guideline OECD 408 (2018) ecommenda ions: 14, 28, 55 mg/kg/day. Ra s (10/sex/g oup) we e o ally adminis e ed he selec ed doses and he con ol g oup ecei ed only he ehicle (po k gela ine). This s udy was pe o med a he Cen al Se ice o Expe imen al Animals om he Uni e si y o Co doba (SAE, Co doba, Spain) in which all animals ecei ed human ca e in acco dance wi h he guidelines o he p o ec ion o animals used o he science 6 pu poses (Di ec i e, 2010/63 EU, Decision, 2012/707/UE, and RD 53/2013). All p ocedu es ha e been app o ed by he E hical Animal Expe imen a ion Commi ee o he Uni e si y o Có doba and by he Jun a de Andalucía (p ojec nº 20/10/2015-348). 2.4. Clinical obse a ions Each animal was obse ed wice daily o mo bidi y and mo ali y and once daily o clinical signs, such as changes in skin, u , eyes o mucous memb anes; sec e ions; changes in gai , pos u e, o handling esponse; abno mal, clonic, o onic mo emen s, and s e eo ypes o biza e beha io . Oph halmic examina ions we e pe o med on all animals be o e ini ia ing he s udy and in he con ol and in he highes dose g oup a he end o ea men . 2.5. Body weigh , ood and wa e consump ion These h ee pa ame e s we e checked weekly in o de o a oid s ess. The mean body weigh pe g oup and sex we e calcula ed weekly and p io o nec opsy om indi idual animals’ da a, as well as he ood and wa e consump ion. The o al ood consumed pe cage was eco ded and he weekly mean in ake pe a was calcula ed. The eed con e sion e iciency (FCE) a io was de e mined acco ding o Escoba e al., (2015) by he a io o ood in ake (g)/ weigh gained (g). 2.6. Hema ology and Biochemis y Blood samples we e ex ac ed om he hea by an in aca diac injec ion unde ligh iso luo ane anes hesia a week 13. Then, he hema ological pa ame e s we e 13 The esul s ob ained ag ee wi h hose o Mellado e al. (2016b) who e alua ed he sa e y o P oallium AP®, an Allium-based comme cial p oduc in a 90-days eeding s udy wi h a s. PTSO is ac ually he majo o ganosul u compound p esen in P oallium AP® (14.5%). Simila ly, nei he clinical signs no any o he changes on gene al, biochemical, hema ological o his opa hological pa ame e s we e de ec ed, and he au ho s de i ed a NOAEL highe han 400 mg/Kg/day a he condi ions assayed. Bo h s udies show a good co ela ion as in he p esen s udy he es i em had a 7- old highe con en o PTSO han P oallium® AP (100 e sus 14.5%) and he NOAEL de i ed o P oallium was 7- old highe . This sugges s ha PTSO has an impo an ole on he oxici y o Allium ex ac s. Ac ually, a MTD o 55 mg/kg in a s o PTSO has been es ablished as p e iously indica ed. And highe doses es ed acco ding o he OECD 425 guideline (2000 mg/kg and 175 mg/kg) esul ed in he dea h o he animal and e iden hepa o oxici y. On he con a y, a single dose o 55 mg/kg did no induce ema kable damage (Llana-Ruiz-Cabello e al, 2015). Mellado-Ga cía e al. (2016a) obse ed ha in a s ea ed wi h 55 mg/kg (3 doses a 0, 24 and 45h and eu hanized a 48h) an inc ease in he glycogen s o age was no iceable in he li e and also a sligh degene a i e p ocess in he chie cells o he s omach. In his ega d, a he dose le els assayed, his opa hological lesions we e absen . This could be explained by he exposu e way employed in his s udy, using gela in as ehicle and wi h he eed. In he p e ious ials, o al ga age wi h a s omach ube a e a as ing pe iod was used ollowing he ecommenda ions o he co esponding OCDE guidelines. The bolus could ha e a mo e dele e ious e ec on he gas oin es inal sys em as he e is a di ec con ac . Di e ences in oxici y be ween PTSO and P oallium AP® could be explained as he oxici y shown by componen s o an essen ial oil can be modula ed by he o he 14 cons i uen s by syne gis ic/an agonis ic phenomena (Escoba e al., 2015; Pa lidou e al., 2004). The in e es o PTSO in compa ison o P oallium® is based on i s di e en chemical p ope ies (highe hyd ophilia) and in i s highe e iciency on i s an ioxidan and an imic obial p ope ies. O he au ho s ha e shown he po en ial oxici y o aqueous ex ac s o di e en medicinal plan s, including Allium sa i um, in Wis a a s. Thus, Sulaiman e al. (2014) adminis e ed o ally o he animals 10 mg/kg o A. sa i um ex ac o 30 days and obse ed al e a ion in he ac i i ies o ma ke enzymes: AST inc eased in li e , kidney and hea , ALT in se um and li e , and ALP ac i i y was educed in se um, hea , kidney and li e . They concluded ha cau ion was equi ed in using un e ined ex ac s o hese he bs in adi ional se ings. The e a e sca ce in i o oxici y da a ega ding o he OSC. Thus, Guyonne e al. (2000) demons a ed he e ec s o some o hem (DAS, DADS, dip opylsul iide (DPS) and dip opyl disul ide (DPDS)) on he ac i a ion o se e al mu agens in male Wis a a s exposed o 1 mmol/kg by ga age o 4 days. They explained he esul s based on he induc ion o cy och ome (CYP) and phase II enzymes ac i i ies. This e ec , he al e a ion o CYP ac i i y, was poin ed ou by o he au ho s as well (Da enpo and Wa go ich, 2005). Mo eo e , hey obse ed hepa o oxici y induced by DAS (bile duc obs uc ion, hype p oli e a ion and ocal poin s o nec osis) in a s ga aged daily wi h 200 mg/kg o 1, 4, o 8 weeks. On he con a y, 8 weeks o exposu e o lowe doses (50 and 100 mg/kg) did no induced li e his opha ological damage. This sugges ha li e could be he a ge o gan o OSC as bo h, DAS (Da enpo and Wa go ich, 2005) and PTSO (Llana-Ruiz-Cabello e al, 2015), ha e shown li e oxici y when a h eshold dose is exceeded. Also, Wu e al. (2001) exposed a s o ally o ga lic oil (GO, 200 mg/Kg) and 3 allyl compounds, DAS (20 and 80 mg/kg), DADS (80 mg/kg), and diallyl 15 isul ide (DATS, 70 mg/kg) 3 imes a week o 6 weeks and examined he an ioxida ion sys em in a li e s and ed blood cells. They ound ha GO, DADS and DATS signi ican ly induced he glu a hione con en (GSH) in blood cells bu nei he GO no any o i s OSC a ec ed he GSH- ela ed an ioxidan enzymes. Hepa ic GSH was no in luenced by ga lic componen s. Bu DADS and DATS signi ican ly inc eased he ac i i y o GSH- educ ase and GSH- ans e ase and dec eased GSH pe oxidase. In he p esen s udy he hema ological pa ame e s we e no in luenced by he PTSO exposu e and sca ce scien i ic da a dealing wi h PSTO a e a ailable o compa e. 5. Conclusions In conclusion, he esul s ob ained con i m he al eady epo ed sa e y p o ile o PTSO o some ood applica ions a he condi ions conside ed. Thus, PTSO did no p omo e oxic e ec s as seen om body weigh changes, ood and wa e consump ion, eed con e sion e iciency, biochemical and blood pa ame e s as well as o gan oxici y and his ological examina ions o main o gans ha could e en ually be a ec ed by i s subch onic adminis a ion (90 days). NOAEL was es ima ed o be ≥ 55 mg/Kg/day. Con lic o In e es The au ho s decla e ha he e a e no con lic s o in e es . Acknowledgemen s The au ho s would like o acknowledge he Spanish Minis e io de Ciencia e Inno ación (P ojec RTC-2017-6199-2), and o he Jun a de Andalucía (P ojec AT 2017-5323) o i s inancial suppo . 16 17 Re e ences Benkeblia, N., Lanzo i, V. (2007). Allium hiosul ina es: chemis y, biological p ope ies and hei po en ial u iliza ion in ood p ese a ion. Food 1, 193–201. Da enpo , D.M., Wa go ich, M.J. (2005). Modula ion o cy och ome P450 enzymes by o ganosul u compounds om ga lic. Food Chem. Toxicol. 43, 1753- 1762. doi: 10.1016/j. c .2005.05.018. Debiagi, F., Kobayashi R.K.T., Nakaza o, G., Panagio, L.A., Mali, S. (2014). Biodeg adable ac i e packaging based on cassa a bagasse, poly inylalcohol and essen ial oils. Ind. C op. P od. 52, 664-670. doi.o g/10.1016/j.indc op.2013.11.032 Eu opean Food Sa e y Au ho i y, 2008. 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The immunomodula o y p ope ies o p opyl-p opane hiosul ona e con ibu e o i s in es inal an i-in lamma o y e ec in expe imen al coli is. Mol. Nu . Food Res. 63(5):e1800653. doi: 10.1002/mn .201800653. Wu, C.C., Sheen, L.Y., Chen, H.W., Tsai, S.J., Lii, C.K. (2001). E ec s o o ganosul u compounds om ga lic oil on he an ioxida ion sys em in a li e and ed blood cells. Food. Chem. Toxicol. 39, 633-569. doi.o g/10.1016/S0278- 6915(00)00171-X RBC: E y h ocy e coun ; HGB: hemoglobin; HCT: hema oc i ; MCV: mean co puscula olume; MCH: mean co puscula hemoglobin; MCHC: mean co puscula hemoglobin concen a ion; PLT: pla ele ( h ombocy e) coun ; RDW: ed blood cell dis ibu ion wid h; T PRO: p o h ombin ime; T CEF: cephalin ime. F: S a is ics ANOVA es ; K.W: K uskal-WallisS a is ic; N.S.: No Signi ican ; &Signi ican ly di e en in compa ison o g oup 4 when p<0.05; # signi ican ly di e en in compa ison o g oup 3 when p<0.05. Table 3 WBC: o al leukocy e coun ; NE: neu ophil; LY: lymphocy e; MO: monocy e; EO: eosinophil; BA: basophil; F: S a is ics ANOVA es ; K.W: K uskal-Wallis S a is ic; N.S.: No Signi ican . DIFFERENTIAL WHITE BLOOD CELLS COUNT DATA SUMMARY MALE FEMALE G oup 1 G oup 2 G oup 3 G oup 4 G oup 1 G oup 2 G oup 3 G oup 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 WBC MEAN 11.43 11.84 12.03 11.70 7.19 6.02 6.83 4.54 103/µL ST. DEV. 3.38 1.70 1.88 3.80 3.67 1.93 2.16 2.70 KW= 0.60 p=0.90; N.S. F(34.3)=1.86 p=0.16; N.S. NE MEAN 16.43 14.59 16.07 16.95 30.24 31.03 24.66 24.99 % ST. DEV. 5.07 2.80 3.53 5.88 27.30 15.46 15.28 11.31 KW=1.47 p=0.69; N.S. F(34.3)=0.35 p=0.79; N.S. LY MEAN 71.87 80.02 78.09 74.52 64.73 62.00 68.32 67.00 % ST. DEV. 14.48 4.25 5.78 8.01 26.04 17.24 14.42 12.00 KW= 5.07 p=0.17; N.S. F(34.3)=0.25 p=0.86; N.S. MO MEAN 3.01 1.52 2.87 2.94 1.44 1.07 0.75 1.57 % ST. DEV. 2.24 1.81 2.33 2.16 1.66 0.74 0.77 1.78 KW=2.71 p=0.44; N.S. KW=2.22 p=0.53; N.S. EO MEAN 3.38 3.22 2.03 4.58 3.17 6.33 4.52 6.94 % ST. DEV. 2.33 1.69 0.58 2.88 2.52 4.53 2.90 5.23 KW=6.70 p=0.08; N.S. KW=3.14 p=0.37; N.S. BA MEAN 0.57 0.70 0.95 1.07 0.41 0.54 0.80 0.51 % ST. DEV. 0.50 0.44 1.39 1.09 0.41 0.41 0.65 0.39 KW=1.42p=0.70; N.S. F(34.3)= 1.01 p=0.40; N.S. Table 3 Table 4 CLINICAL BIOCHEMISTRY DATA SUMMARY MALE FEMALE G oup 1 G oup 2 G oup 3 G oup 4 G oup 1 G oup 2 G oup 3 G oup 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 GLUC MEAN 143.50 146.50 128.40 123.60 125.60 121.60 105.20 104.00 mg/dL ST. DEV. 22.73 27.44 26.31 20.30 16.64 17.20 27.59 44.93 F(36.3)=2.12 p=0.12; N.S. KW=4.90 p=0.18; N.S. UREA MEAN 35.22 31.67 33.99 35.37 30.26 31.87 34.90 34.22 mg/dl ST. DEV. 4.83 2.95 4.37 3.16 4.63 3.36 7.02 4.14 F(36.3)=1.92 p=0.14; N.S. F(36.3)=1.85 p=0.16; N.S. CREAT MEAN 0.30 0.28 0.29 0.29 0.34 0.35 0.35 0.34 mg/dL ST. DEV. 0.04 0.02 0.03 0.03 0.06 0.03 0.07 0.02 F(36.3)=0.48 p=0.70; N.S. F(36.3)=0.26 p=0.86; N.S. BILI-T MEAN 0.21 0.31 0.21 0.22 0.26 0.28 0.25 0.26 mg/dL ST. DEV. 0.02 0.32 0.02 0.03 0.03 0.02 0.03 0.01 KW=1.41 p=0.70; N.S. F(36.3)=2.15 p=0.11; N.S. CHOL MEAN 107.50 95.40 93.50 88.50** 104.20 101.80 112.40 99.20 mg/dL ST. DEV. 13.83 15.36 10.60 8.50 11.12 20.75 20.58 15.36 F(36.3)=4.25 p<0.01** F(36.3)=1.08 p=0.36; N.S. TRIGL MEAN 156.40 163.40* 131.80 123.00 78.60 104.80 84.40 77.80 mg/dL ST. DEV. 33.81 36.16 27.59 20.96 18.87 28.06 21.53 16.94 KW=10.48 p<0.05* F(36.3)=1.85 p=0.16; N.S. AST MEAN 147.52 145.76 135.39 183.73 264.52 246.91 251.94 264.62 U/L ST. DEV. 17.74 26.66 17.66 101.03 94.90 92.11 73.01 122.10 KW=3.44 p=0.33; N.S. KW= 0.39 p=0.95; N.S. ALT MEAN 30.46 38.36 33.42 31.44 38.31 35.40 37.03 35.22 U/L ST. DEV. 6.26 16.86 6.09 2.42 10.34 17.81 5.26 10.55 KW=3.12 p=0.38; N.S. KW=2.41 p=0.49; N.S. ALKP MEAN 91.20 98.20 88.30 89.60 62.00 62.90 66.40 60.60 U/L ST. DEV. 9.62 18.50 26.97 15.21 12.14 9.99 17.22 9.13 Table 4 GLUC: glucose; CREAT: c ea inine; Bili-T: Bili ubin, o al; CHOL:choles e ol, o al; TRIGL: iglyce ides; AST:aspa a e amino ans e ase; ALT: alanine amino ans e ase; ALKP: alkaline phospha ase; ALB:albumin; TOT PROT: p o ein, o al; Na+:sodium; K+:po assium; Ca++: calcium. F: S a is ics ANOVA es ; K.W:K uskal-WallisS a is ic; N.S.: No Signi ican *Signi ican ly di e en om con ol. *when p<0.01 **Signi ican ly di e en om con ol. **when p<0.01 KW=0.56 p=0.65; N.S. F(36.3)=0.39 p=0.76; N.S. ALB MEAN 3.93 3.88 3.98 4.00 4.80 4.59 4.66 4.72 g/dl ST. DEV. 0.47 0.25 0.32 0.29 0.35 0.34 0.59 0.34 KW=1.54 p=0.67; N.S. F(36.3)=0.47 p=0.70; N.S. TOT PROT MEAN 6.24 6.26 6.26 6.20 6.46 6.78 6.78 6.54 g/dl ST. DEV. 0.34 0.48 0.37 0.29 0.44 0.35 0.68 0.51 F(36.3)=0.06 p=0.98; N.S. F(36.3)=1.05 p=0.38; N.S. Na+ MEAN 134.80 133.70 133.60 132.80 142.60 139.60 140.20 139.40 mmol/L ST. DEV. 10.40 3.59 3.24 4.08 4.62 7.12 11.64 7.78 KW=2.00 p=0.57; N.S. KW=1.65 p=0.65; N.S. K+ MEAN 7.87 8.89 7.90 8.38 9.68 12.81 14.93 10.34 mmol/L ST. DEV. 1.58 1.62 0.77 1.29 6.97 6.18 7.61 5.67 F(36.3)=1.26 p=0.30; N.S. KW=6.35 p=0.10; N.S. Ca++ MEAN 10.87 10.28 10.82 10.51 11.30 11.42 10.76 11.25 mg/dL ST. DEV. 1.04 0.66 0.64 0.47 1.06 1.26 0.87 1.36 KW=4.43 p=0.22; N.S. KW=2.34 p=0.51; N.S. Table 5 F: S a is ics ANOVA es ; K.W: K uskal-Wallis S a is ic; N.S.: No Signi ican . * Signi ican ly di e en om g oup 3 in compa ison o g oup 1 when p<0.05. ORGAN WEIGHT DATA SUMMARY MALE FEMALE G oup 1 G oup 2 G oup 3 G oup 4 G oup 1 G oup 2 G oup 3 G oup 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 BODY W. MEAN 633.80 623.10 611.5 625.90 BODY W. MEAN 326.80 338.00 321.10 321.30 (g) ST. DEV. 38.25 28.71 26.50 48.14 (g) ST. DEV. 11.50 29.08 23.81 18.31 F(36.3)=0.19 p=0.90; N.S. F(36.3)=1.34 p=0.29; N.S. BRAIN MEAN 2.09 2.13 2.12 2.07 BRAIN MEAN 1.96 2.03 2.01 2.06 (g) ST. DEV. 0.21 0.14 0.17 0.23 (g) ST. DEV. 0.21 0.10 0.12 0.15 KW=0.09 p=0.99; N.S. KW=2.35 p=0.50; N.S. LIVER MEAN 21.06 18.49 18.40 18.41 LIVER MEAN 8.41 9.54 8.90 8.48 (g) ST. DEV. 4.86 1.45 1.77 2.40 (g) ST. DEV. 1.36 1.09 1.08 0.83 F(36.3)=1.99 p=0.13; N.S. F(36.3)=2.21 p=0.10; N.S. HEART MEAN 2.14 1.98 1.82* 1.99 HEART MEAN 1.21 1.28 1.20 1.22 (g) ST. DEV. 0.18 0.19 0.19 0.32 (g) ST. DEV. 0.13 0.13 0.14 0.16 F(36.3)=3.11 *p<0.05 F(36.3)=0.67 p=0.59; N.S. SPLEEN MEAN 1.17 1.16 1.16 1.45 SPLEEN MEAN 0.80 0.86 0.74 0.75 (g) ST. DEV. 0.10 0.15 0.14 0.93 (g) ST. DEV. 0.11 0.19 0.07 0.09 KW=0.2426 p=0.9704; N.S. F(36.3)=1.71 p=0.18; N.S. KIDNEYS MEAN 4.05 4.10 3.99 4.06 KIDNEYS MEAN 2.13 2.30 2.15 2.06 (g) ST. DEV. 0.30 0.41 0.37 0.28 (g) ST. DEV. 0.23 0.23 0.20 0.14 F(36.3)=0.17 p=0.92; N.S. F(36.3)=2.48 p=0.08; N.S. THYMUS MEAN 0.88 0.80 0.83 0.71 THYMUS MEAN 0.67 0.70 0.66 0.60 (g) ST. DEV. 0.25 0.12 0.15 0.27 (g) ST. DEV. 0.08 0.13 0.20 0.10 F(36.3)=1.20 p=0.32; N.S. F(36.3)=1.02 p=0.40; N.S. TESTES MEAN 3.92 3.95 3.77 3.83 UTE./CERV. MEAN 0.87 0.76 1.03 0.94 (g) ST. DEV. 0.44 0.29 0.32 0.28 (g) ST. DEV. 0.24 0.17 0.81 0.32 F(36.3)=0.62 p=0.61; N.S. KW=1.32 p=0.73; N.S. EPIDIDIMS MEAN 1.88 2.05 2.36 2.07 OVARIES MEAN 0.26 0.31 0.23 0.25 (g) ST. DEV. 0.24 0.52 0.98 0.28 (g) ST. DEV. 0.0.8 0.10 0.06 0.07 KW=4.00 p=0.26; N.S. F(36.3)= 1.90 p=0.15; N.S. ADRENALS MEAN 0.11 0.13 0.12 0.12 ADRENALS MEAN 0.15 0.12 0.10 0.12 (g) ST. DEV. 0.04 0.05 0.07 0.05 (g) ST. DEV. 0.08 0.04 0.02 0.08 KW=0.72 p=0.87; N.S. KW=4.95 p=0.18; N.S. Table 5 Table 6 F: S a is ics ANOVA es ; K.W: K uskal-Wallis S a is ic; N.S.: No Signi ican . ORGAN WEIGHT/BODY WEIGHT RATIO DATA SUMMARY MALE FEMALE G oup 1 G oup 2 G oup 3 G oup 4 G oup 1 G oup 2 G oup 3 G oup 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 BRAIN MEAN 0.33 0.34 0.36 0.33 BRAIN MEAN 0.60 0.60 0.63 0.64 (%) ST. DEV. 0.03 0.03 0.06 0.04 (%) ST. DEV. 0.07 0.05 0.6 0.05 KW=4.99 p=0.17; N.S. F(36.3)=1.17 p=0.34; N.S. LIVER MEAN 3.31 2.97 3.22 2.93 LIVER MEAN 2.58 2.82 0.77 2.64 (%) ST. DEV. 0.71 0.15 0.71 0.21 (%) ST. DEV. 0.42 0.23 0.27 0.24 KW=2.03 p=0.57; N.S. F(36.3)=1.47 p=0.24; N.S. HEART MEAN 0.33 0.31 0.32 0.32 HEART MEAN 0.37 0.38 0.38 0.38 (%) ST. DEV. 0.04 0.02 0.05 0.05 (%) ST. DEV. 0.04 0.05 0.06 0.05 F(36.3)=0.64 p=0.59; N.S. KW=1.12 p=0.77; N.S. SPLEEN MEAN 0.18 0.19 0.20 0.23 SPLEEN MEAN 0.25 0.25 0.23 0.23 (%) ST. DEV. 0.02 0.02 0.03 0.14 (%) ST. DEV. 0.03 0.05 0.02 0.03 KW=2.15 p=0.54; N.S. F(36.3)=0.76 p=0.52; N.S. KIDNEYS MEAN 0.63 0.65 0.70 0.65 KIDNEYS MEAN 0.65 0.68 0.67 0.64 (%) ST. DEV. 0.05 0.05 0.15 0.06 (%) ST. DEV. 0.07 0.06 0.06 0.06 KW=1.42 p=0.70; N.S. KW=2.53 p=0.47; N.S. THYMUS MEAN 0.14 0.13 0.15 0.11 THYMUS MEAN 0.21 0.21 0.21 0.19 (%) ST. DEV. 0.02 0.02 0.05 0.04 (%) ST. DEV. 0.03 0.04 0.06 0.03 KW=4.80 p=0.19; N.S. F(36.3)=0.68 p=0.57; N.S. TESTES MEAN 0.62 0.63 0.66 0.61 UTE./CERV. MEAN 0.27 0.23 0.32 0.29 (%) ST. DEV. 0.07 0.05 0.14 0.07 (%) ST. DEV. 0.07 0.05 0.25 0.10 KW=1.16 p=0.76; N.S. KW=2.47 p=0.48; N.S. EPIDIDIMS MEAN 0.30 0.32 0.41 0.33 OVARIES MEAN 0.08 0.09 0.07 0.08 (%) ST. DEV. 0.04 0.08 0.18 0.05 (%) ST. DEV. 0.02 0.03 0.02 0.02 KW=5.544 p=0.1361; N.S. KW=3.53 p=0.32; N.S. ADRENALS MEAN 0.02 0.02 0.02 0.02 ADRENALS MEAN 0.05 0.04 0.03 0.04 (%) ST. DEV. 0.01 0.01 0.01 0.01 (%) ST. DEV. 0.02 0.01 0.01 0.02 KW=0.45 p=0.93; N.S. KW=4.49 p=0.21; N.S. Table 6 Table 7 F: S a is ics ANOVA es ; K.W: K uskal-Wallis S a is ic; N.S.: No Signi ican . ORGAN WEIGHT/BRAIN WEIGHT RATIO DATA SUMMARY MALE FEMALE G oup 1 G oup 2 G oup 3 G oup 4 G oup 1 G oup 2 G oup 3 G oup 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 LIVER MEAN 1011.66 874.10 876.04 892.82 LIVER MEAN 437.12 470.10 444.59 411.74 (%) ST. DEV. 229.58 103.29 131.13 92.16 (%) ST. DEV. 104.65 48.18 56.83 41.47 KW=4.08 p=0.25; N.S. F(36.3)=1.27 p=0.30; N.S. HEART MEAN 103.79 93.72 86.79 97.94 HEART MEAN 62.93 63.41 60.00 59.58 (%) ST. DEV. 16.99 12.97 10.89 23.79 (%) ST. DEV. 13.12 8.56 8.83 9.81 KW=6.19 p=0.10; N.S. F(36.3)=0.37 p=0.78; N.S. SPLEEN MEAN 56.36 54.78 55.10 55.42 SPLEEN MEAN 41.83 42.21 37.10 36.61 (%) ST. DEV. 7.85 6.83 7.14 7.75 (%) ST. DEV. 9.96 9.33 3.40 6.17 F(36.3)=0.084 p=0.97; N.S. F(36.3)=1.52 p=0.23; N.S. KIDNEYS MEAN 195.30 193.90 190.21 198.22 KIDNEYS MEAN 110.18 113.37 107.39 100.25 (%) ST. DEV. 22.29 27.64 30.06 26.91 (%) ST. DEV. 18.71 12.13 13.40 12.68 F(36.3)=0.15 p=0.93; N.S. KW=5.94 p=0.11; N.S. THYMUS MEAN 42.63 37.87 39.25 34.33 THYMUS MEAN 34.57 34.51 33.29 28.27 (%) ST. DEV. 13.08 6.61 7.85 12.04 (%) ST. DEV. 5.25 5.96 12.14 4.26 F(36.3)=1.13 p=0.35; N.S. KW=5.75 p=0.12; N.S. TESTES MEAN 189.99 187.00 179.45 187.33 UTE./CERV. MEAN 44.81 37.40 51.51 46.40 (%) ST. DEV. 33.59 23.50 24.34 28.69 (%) ST. DEV. 12.42 8.40 40.13 18.00 F(36.3)=0.26 p=0.85; N.S. KW=2.62 p=0.45; N.S. EPIDIDIMS MEAN 89.92 96.89 112.09 100.46 OVARIES MEAN 13.04 15.32 11.31 12.10 (%) ST. DEV. 7.49 24.61 46.97 12.46 (%) ST. DEV. 3.734 5.41 3.20 3.43 KW=3.96 p=0.27; N.S. F(36.3)=1.85 p=0.16; N.S. ADRENALS MEAN 5.42 5.40 5.91 5.75 ADRENALS MEAN 8.00 6.10 4.82 5.98 (%) ST. DEV. 2.59 2.83 3.99 2.77 (%) ST. DEV. 4.87 2.02 1.33 3.97 KW=0.50 p=0.92; N.S. KW=4.27 p=0.23; N.S. Table 7 Figu e 1 Click he e o access/download;Figu e; igu a 1. ajo. i Figu e 2 Click he e o access/download;Figu e;Figu e 2. Body weigh . i Figu e 3 Click he e o access/download;Figu e;Figu e 3. Mean body weigh gain. i