Ph ton. The Journal of Toxicology and Health. Subchronic toxicological effects of pea protein isolate (nutralys) on wistar rats: A ninety-day dietary

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1 he Journal of oxicology and Health. Photon ( - Original Research Article. ISJN: - he Journal of oxicology and Health Ph ton Subchronic toxicological effects of pea protein isolate (nutralys on wistar rats: A ninety-day dietary Chentouf Aouatif a, Philippe Looten a, Srinivasan M b*, Srinivas A b, Yogeshkumar V. Murkunde b a Biology and Nutrition Department, oxicology Service, Roquette Freres, Lestrem, France b International Institute of Biotechnology and oxicology (IIBA, Padappai-, amil Nadu, India Article history: Received: March, Accepted: March, Available online: August, Keywords: Pea Protein Isolate (Nutralys, subchronic toxicity, dietary,, NOAEL Abbreviations: OECD: Organization for Economic Co-operation and Development, No Observed Adverse Effect Level (NOAE Corresponding Author: Dr. Srinivasan M.* Senior Scientist sini@sify.com Phone: (o + - Dr. Chentouf Aouatif Senior Scientist aouatif.chentouf@roquette.com Phone: (o ( Philippe Looten oxicologist Phone: (o ( Dr. Srinivas A. Senior Scientist srinivas@iibat.com Phone: (o + -. Introduction Pea Protein Isolate (Nutralys is a water extract of dry pea (Pisum Sativum manufactured by Roquette in our Vic sur Aisne plant (located in Northern Paris. his product is spray-dried and granulated to ensure easy blending and use in food processing and is widely used for maintaining an ideal body weight. Proteins are nitrogen- containing substances that are formed by amino acids. hey serve as the major structural component of muscle and other tissues in the body. In addition, they are used to produce hormones, enzymes and hemoglobin. Proteins can also Dr. Yogeshkumar V. Murkunde HOD Yogeshkumar@iibat.com Phone: (o + - Abstract Pea protein isolate is a derivative of vegetable that has been used as a nutrient in foods. Since no information has been published about the safety of Pea protein isolate (Food Grade by Roquette Frères, a -day feeding study was conducted, using Wistar rats of either sex as experimental animals. Rats fed with dietary levels of low ( ppm, intermediate ( ppm and high ( ppm Pea protein isolate for days did not reveal any induced toxicological changes as their clinical signs, body weights, food consumption, water consumption, hematological, blood biochemical and urinalysis were comparable with concurrent control animals. Further, organ weights, gross and histological examinations did not reveal any systemic toxicity induced by Pea Protein consumption. aken together, under the current experimental conditions, the oral diet No Observed Adverse Effect Level (NOAE of Pea Protein Isolate for males and female rats were found to be % (equivalent to mg/kg b.w. for male rats and mg/kg b.w. for female rats respectively. Citation: Aouatif C., Looten P., Srinivasan M., Srinivas A., Murkunde Y.V.,. Subchronic toxicological effects of pea protein isolate (nutralys on wistar rats: A ninety-day dietary. he Journal of oxicology and Health. Photon, -. be used for energy; however, they are not the primary choice as an energy source. For proteins to be used by the body, they need to be metabolized into their simplest form, amino acids. here have been amino acids identified that are needed for human growth and metabolism. welve of these amino acids (eleven in children are termed nonessential, meaning that they can be synthesized by our body and do not need to be consumed in the diet (Hoffman and Michael,. he remaining amino acids cannot be synthesized in the body and are described as essential: Ph ton

2 meaning that they need to be consumed in our diets. he absence of any of these amino acids will compromise the ability of tissue growth, or its ability to repair or maintain. Pea Protein Isolate is composed of all essential amino acids and is recommended as a dietary supplement to compensate the daily protein requirements of the human body. As like any other commercial dietary product, extensive toxicological potential of the respective product needs to be evaluated before its use by humans. Hence in the current study, we have assessed the sub chronic toxic effect of Pea Protein Isolate when administered as a diet supplement for a period of days in Wistar rats as animal model. his study was conducted as per OECD est Guidelines number. Prior approval of Institutional Animal Ethics Committee (IAEC was obtained before the commencement of these in vivo studies at International Institute of Biotechnology and oxicology (IIBA, Chennai, India.. Materials and Methods he present study was conducted in accordance with the OECD Guideline for the esting of Chemicals, Repeated Dose -day Oral oxicity study in Rodents, (No., Adopted: st September,... Preparation of diet and stability of Pea Protein Isolate (Nutralys, in the diet Pea Protein Isolate (Nutralys manufactured and supplied by Roquette Freres, France is a high quality white powder source food grade with % Pea Protein content, extracted in water. o administer this Pea Protein via diet, it was minced to fine powder and mixed with Standard gamma irradiated diet supplied by M/s. etragon Chemie Pvt. Ltd., Bangalore, India, to the final feed formulations of., and w/w%. he stability of this pea protein in diet was examined periodically throughout the duration of the study. he Pea Protein Isolate (Nutralys, has shown to be very stable in the formulated diet. Homogeneity of Pea protein isolate (Nutralys, in the diet was found to be % and was maintained throughout the duration of the study... Animal husbandry One hundred and twenty healthy Wistar (Crl: WI rats of either sex aged - weeks were obtained from the animal house facility, IIBA during the year. Females used in the study were nulliparous and non pregnant. Animals were allowed to adapt to the animal room conditions for a week before the initiation of the study. Every five animals of the same sex were housed in stainless steel cages under controlled environmental conditions (temperature ±. C, humidity ±% and a h light/dark cycle. Pea Protein mixed diet and reverse osmosis water were available ad libitum... Objective of Research o find the sub chronic dietary toxicity (NOAE of the Pea Protein Isolate (Nutralys in Wistar rats and according to the OECD est Guideline and understand the mechanisms of toxicity if any, induced by Pea Protein Isolate (Nutralys... Study Design After acclimation, animals were randomly distributed, into six groups (consisting of males and females per group namely G - control, G - Low ( ppm, G - Intermediate ( ppm, G - ( ppm, G - control and G - high ( ppm. he test substance was administered daily by admixture with the diet for a period of days to the G, G, G and G animal groups. group (G and satellite control group (G of rats were similarly treated but without the test substance. Rats in satellite groups were given only diet without the test item for an additional days to evaluate any possible withdrawal effects. hey were individually observed once daily for clinical signs. Body weight of the animals was measured once weekly. Food intake and water of the animals were measured once daily and reported weekly... Hematological and biochemical Examination Following a ninety-day feeding, the animals were fasted overnight for h prior to the scheduled blood collection. hey were anesthetized with CO and about ml of blood was collected per animal from the retro-orbital venous plexus into pre-labelled EDA and heparinized vials for hematological and biochemical analysis respectively. Hematology analysis includes erythrocytes, hemoglobin, hematocrit, mean cell volume (MCV, mean carpuscular hemoglobin (MCH, mean carpuscular haemoglobin concentration (MCHC, platelet count, leukocytes and leukocyte differential count analyzed by using fully automated hematology analyzer (Advia, Siemens, USA. Prothrombin time was done by hromborel-s method and clotting time was done by capillary tube method. Ph ton

3 Heparinized plasma used for biochemical analysis was obtained by centrifugation at x g for min. Biochemical analysis includes glucose, urea, creatinine, total cholesterol, triglycerides, aspartate transaminase (AS, alanine transaminase (AL, alkaline phosphatase (ALP, total protein, albumin, calcium and phosphorus analyzed by using fully automated biochemistry analyzer (Dimension Xpand plus, Siemens, USA. Serum electrolytes (sodium and potassium were assayed using electrolyte analyzer (Humalyte, Human GmbH and Germany... Gross and Histopathology At autopsy, the liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, thymus, spleen, brain and heart were removed and weighed. In addition, the liver, spleen, lungs, heart, aorta, kidneys, adrenals, brain, pituitary, trachea, thyroid, parathyroid, oesophagus, small intestine, large intestine, salivary glands, lymph nodes (Mandibular and mesenteric, spinal cord, thymus, stomach, pancreas, ovaries, uterus, accessory sex organs, female mammary gland, prostate, urinary bladder, peripheral nerve (Sciatic, bone marrow (sternum, skin were collected from all animals and preserved in % neutral buffered formalin, except testis, which was preserved in Modified Davidson s for hours and transferred in to % buffered formalin after minutes washing in running tap water. he lungs were inflated with % neutral buffered formalin before preservation. he organs were processed in vacuum infiltration tissue processor (Sakura issue-ek VIP M, Japan, embedded (Sakura issue-ek EC M, Japan with paraffin wax, sectioned (Microm GmbH, Germany at approximately µm thick, stained (Leica S Autostainer, Germany with hematoxylin and eosin, evaluated through light microscopy (Nikon i, Japan and images were captured through an image analyzer (Q Imaging systems, Canada. he identity and analysis of the pathology slides were blind to the pathologist... Statistical methods Body weight, food consumption, hematology, clinical biochemistry and organ weights were subjected for Modified-levene equal-variance test for homogeneity. Homogenous data was submitted to Analysis of Variance (ANOVA followed by Student-Newman-Keul s test for post hoc comparison. If the data is heterogeneous it was subjected to nonparametric tests (NCSS, statistical software. Differences were considered to be significant when P value was less than... Results and Discussion he aim of the present study was to evaluate the safety of Pea Protein Isolate (Nutralys using a sub chronic toxicity study design. he in vivo toxicity studies are usually divided into four categories: acute, subacute, subchronic and chronic. Acute exposure is defined as exposure to a chemical for less than h and usually used for labeling and classification purpose. Subacute and sub chronic toxicity studies refers to repeated exposure to a chemical for in rodent or non rodent species generally for a period of days, days and more than three months respectively. hese studies are usually performed to attain NOAEL concentrations of the test compound at the respective duration period. Lack of sub chronic toxicity information of Pea Protein Isolate, which is used as a part of food supplement, stimulated us to conduct the current study in Wistar rats by oral route. Data obtained from this study can contribute substantially to the scientific fraternity, to understand the toxicity mechanisms of Pea Protein if any. he OECD Guidelines for esting of Chemicals, Section ( and United States Environmental Protection Agency Health Effects est Guidelines are preferred the rat as the standard rodent species. We need to evaluate the safety of Pea protein isolate (Nutralys in same species. herefore, a conscientious and careful safety evaluation of Pea protein isolate (Nutralys in rat is necessary. Additionally, there is no subchronic oral toxicity report to show that Pea protein isolate (Nutralys has been studied in rats. he rat has been one of the main mammalian species used in preclinical studies ranging from pharmacology and safety assessment. he use of rats as models in safety evaluations is currently required in international guidelines for both chemicals and pharmaceuticals (Hedrich,. FDA Guidelines for oxicological Principles for the Safety Assessment of Food Ingredients, Redbook, Chapter IV.C..a ( indicated that short- term toxicity studies are conducted with rats and mice. herefore, both male and female rats, which are healthy and have not been subjected to previous experimental procedures, should be used in sub chronic toxicity study. Ph ton

4 able-: Summary of Hematology Parameters Day- Sex: Male RBC Hb HC PL WBC N L E M B LUC C P /µl (% /µl ( /µl (% (% (% (% (% (% (Sec (Sec G G Low G Intermediate G * G G Data are expressed as Mean ± S.D.(n=, ype of analysis- one-way ANOVA test at. level of significance, G- -G- =* represents the statistical difference from G - - to G - Low (p<. In the current study, no mortality or clinical signs of toxicity was observed in any of the treated animals. Body weight gain (Fig. & and feed consumption (Fig. & in pea protein administered animals were comparable with untreated animals. A statistical significant difference was observed in water consumption of high male rats on th week when compared with that in the control group. However, this change was considered to be treatment related (data not shown. Estimation of hematological assays on Day, reveal a significant increase of eosinophils in male rats and prothrombin time in female rats of low group when compared with control animals (able-. Further, significant decrease in platelets and neutrophils and increase in lymphocyte counts were observed in female rats of high group (%-able-. On day, a statistically significant change was observed in prothrombin time of satellite high (G group males (able - and large unstained cells of females (able - when compared with satellite control (G group animals. Biochemical assays on Day, showed a significant increase in triglyceride levels in all the three treated groups of female rats (able - when compared with control animals. However these increased levels of triglycerides were not observed in male rats (able -. animals were bled on day to assess the reversal effects, AS levels in male rats(able - and glucose, urea, BUN, triglycerides and potassium levels in female rats(able - were significantly higher when compared to control animals. he liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, thymus, spleen, brain and heart, and relative weights of these to body weights. he absolute weight of the testes of male rats in the low group was higher when compared to the control group, whereas such an increase was not found in other treated groups. Similarly, absolute weight of the spleen of female rats in the high group was increased significantly when compared to the control group. No significant differences in any of the organs were observed on Day in any treated animals, except a significant decrease of spleen in male rats. Gross pathological examination did not reveal any Pea Protein induced changes in the groups tested of either sex. Further, no adverse histo-pathological findings were observed in any of the treated animals of either sex. Ph ton

5 able-: Summary of Hematology Parameters Day- Sex: Female RBC Hb HC PL WBC N L E M B LUC C P /µl (% /µl ( /µl (% (% (% (% (% (% (Sec (Sec G G Low G Intermediate G G G * *..*.* able - RBC /µ l Hb HC MC V Summary of Hematology Parameters Day- MCH (% (f (pg MCH C ( PL / µl WB C ( / µl N L E M B LU C C (% (% (% (% (% (% (Sec Sex : Male P (Se c G G... * Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =* represents the statistical difference from G - - to G - (p<. Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =* represents the statistical difference from G - - to G - (p< he results of the present study not only provide scientific evidences to evaluate the safety of Pea protein isolate (Nutralys, using a subchronic toxicity study design in rats, but also to find the range of Pea protein isolate (Nutralys, for the subsequent studies in rats, such as carcinogenicity study. Furthermore, this is the first time Pea protein isolate (Nutralys, has been studied in sub chronic oral toxicity in rats. observation that were attributable to Pea protein isolate (Nutralys, ingestion. Feed consumption during the study was found to be normal in wither sex. Further, a gradual increase in body weights were observed in rats fed with Pea Protein throughout the duration of the study. Similar findings were noticed by Mejia., a&b, where rats were fed with cruciferin-rich protein isolate PurateinQ for weeks. (Plass,, he also reported similar findings. During the feeding period, there were no deaths or signs of toxicity on gross Ph ton

6 able - Summary of Hematology Parameters Day- Sex : Female HC MC MC WB RBC Hb MCH PL N L E M B LUC C P V HC C ( /µl (% (f (pg ( (Sec (S /µl (% (% (% (% (% (% /µl ec G * G Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =* represents the statistical difference from G - - to G - (p<. Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =* represents the statistical difference from G - - to G - (p<. able - G G Low G Intermedia te G G G Glu Urea BUN Cre Cho ri d Summary of Clinical Biochemistry Parameters Day- AS (U/ AL (U/ ALP Pro Alb Cal (U/ Ph os GG (U/ Sex : Male * Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =Not significant(p>. G- -G- = Not significant(p>. G- -G- = Not significant(p>. G- -G- =* represents the statistical difference from G -- to G - (p<. Na m ol/ K m ol/. able - Summary of Clinical Biochemistry Parameters Day- Sex : Female Ph ton

7 G G Low G Intermed iate Glu Urea BUN Cre Cho ri AS AL ALP Pro Alb Cal (U/ (U/ (U/ * * ( Ph os GG (U/ Na m ol/ K mol / G G G * * * Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =* represents the statistical difference from G - - to G -Low (p<. G- -G- =* represents the statistical difference from G - - to G - Intemediate (p<. G- -G- =* represents the statistical difference from G - - to G - (p<. G- -G- =* represents the statistical difference from G - - to G - (p< able G G Glu Urea BUN Cre Cho ri g / Summary of Clinical Biochemistry Parameters Day- g / AS (U/L AL (U/ AL P (U/L Pro Alb Cal Phos (L (L GG (U/L Na m ol/ Sex : Male ± ± * ± ± Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =* represents the statistical difference from G - - to G - (p<. Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =* represents the statistical difference from G - - to G - (p<.. K m ol/. Ph ton

8 able Summary of Clinical Biochemistry Parameters Day- Glu Urea BUN Cre Cho ri AS AL ALP Pro Alb Cal d (U/ (U/ (U/ Ph os. G G (U /L. Sex : Female G ± ± G. * * * *. * ± ± Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =* represents the statistical difference from G - - to G - (p<. Data are expressed as Mean ± S.D.(n= ype of analysis- one-way ANOVA test at. level of significance G- -G- =* represents the statistical difference from G - - to G - (p<. Na mo l/. K mo l/. dependent manner. Further, gross and histological examinations of these animals did not reveal any abnormal findings in these organs. Ophthalmoscopic examination and data of the functional observational battery tests did not reveal any neurological toxicity induced by Pea Protein dietary administration. Further, though minor significant changes occurred with a few biochemical assays like, AS, BUN, glucose and triglycerides, these changes were not test compound dependent and overall, Pea Protein Isolate administration in rats did not alter any liver or kidney function. Similarly, no hematological alterations were observed, which suggests that these test compounds when administered as food supplement may not have any adverse effect on hemopoietic system. he absolute organ nor the relative organ weights, in Pea protein isolate (Nutralys, ingested by the rats were normal, except an increase in the absolute weight of the spleen in females and decrease of the testes organ in male rats of the high and low respectively. hese minimal alterations can be attributed to intra animal variation and can not be imparted to test compound induced toxicity as these changes are not consistent in a here were no adverse lesions observed in gross and histological examinations of other vital organs. aken together, as no adverse effects were observed in any of the rats administered with Pea Protein Isolate of different concentrations, NOAEL of this test compound in Wistar rats can be defined as, ppm of diet (equivalent to for male and for female mg/kg b.w/day under the present experimental conditions. Based on our findings, Pea Protein can be considered as non toxic when administered through diet and is recommended for oral consumption as a food supplement. Conflict of interest he authors declared no conflicts of interest. Acknowledgements he authors are grateful to the IIBA management and for Roquette Freres, France supporting for this work. We thank M. Goparaju for his cooperation in statistical analysis and O. Barathkumar for their contribution to the laboratory work.. Author s Contribution and Competing Interests All authors have equally contributed for this work. Ph ton

9 References Grice H C., Heggtveiet H.A.,. he relevance of humans of myocardial lesions induced by rats by marine and rapeseed oils. In: and low erucic rapeseed oils. Production, usage, chemisty and toxicological examinations. (Ed. J.K.G. Kramer, F.D. Sauer & W.J. Pigden Academic Press, oronto, Canada. Hedrich H.J.,(ed (. Genetic monitoring of Inbred Strains of Rats.Gustav Fischer, New York. Hoffman Michael J,.. International Society of Sports Nutrition Journal of Sports Science and Medicine., -. Mejia L.A., b. A -week dietary toxicity study in rats of a napin-rich canola protein isolate. Reg. oxicology & Pharmacology,, -. OECD Guidelines for the esting of Chemicals, No., Adopted: st September,. Plass R.,. oxicological evaluation of rapeseed products in a sub- acute feeding study in rats. Dietary Nahrung :-. Ph ton