A 90-Day Oral Toxicity Study of Tartrazine, a Synthetic Food Dye, In

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A 90-Day Oral Toxicity Study of Tartrazine, a Synthetic Food Dye, In International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 3, Suppl 3, 2011 Research Article A 90­DAY ORAL TOXICITY STUDY OF TARTRAZINE, A SYNTHETIC FOOD DYE, IN WISTAR RATS IMANE HIMRI 1 *, SAID BELLAHCEN 2, FAIZA SOUNA 1, FATIMA BELMEKKI 2, MOHAMMED AZIZ 2, MOHAMED BNOUHAM 2, JOUHAR ZOHEIR 3, ZOLIKHA BERKIA 4, HASSANE MEKHFI 2, ENNOUAMANE SAALAOUI 1 1: Université Mohamed Ier, Faculté des Sciences, Laboratoire de Biochimie, 2 : Université Mohamed Ier, Faculté Des Sciences, Laboratoire De Physiologie Et Ethnopharmacologie, 3 : Laboratoire d’Analyse Zoheir, Oujda, 4 : Cabinet d’Anatomie Pathologique, Oujda Received: 28 Feb 2011, Revised and Accepted: 30 March 2011 ABSTRACT The following study is about Tartrazine (E 102) which is known as an azo dye used in food products, drugs and cosmetics. As a part of the safety assessment of Tartrazine, a 13‐week subchronic oral toxicity study was performed on Wistar rats of both sexes. The animals were divided into 5 groups of 6 animals each, 3 of each sex, and fed a diet containing 5, 7.5, or 10 mg/kg b.w, of Tartrazine and 3.75 mg/kg b.w, of Sulfanilic acid. There were no treatment‐related adverse effects with regard to body weight, food and water consumption. Their blood samples were analyzed for hematological measurements, Glucose, Creatinine, Blood urea nitrogen, Cholesterol total, Triglecerid, alanine aminotransferase, aspartate aminotransferase. The Stomach, Jejunum, Liver, Kidneys tissues were also processed for histological examination. The present study shows that Tartrazine and Sulfanilic acid induced a morphological change from the discoid shape to an echinocytic form in rat RBCs. Relative weights of the liver were significantly increased in group treated with 10 mg/kg b.w, of Tartrazine. Our data showed a significant increase in GLU, CREA, CHOL, TG, AST, and total Protein in serum of rats treated with Tartrazine and Sulfanilic acid compared to control rats and these significant changes were more apparent in high doses than low ones. The histopathological changes of Liver and Kidney were in accordance with the biochemical findings. Keywords: Tartrazine, Subchronic toxicity, Hepatotoxicity, Nephrotoxicity, Wistar rats. INTRODUCTION The study of the carcinogenetic and mutagenetic effects of Tartrazine were established by some authors which gives variable Food additives are products added to the basic foodstuffs with an results 6‐16. aim of improving its aspect, savour, taste, colour, texture, food value and conservation. When the Tartrazine reaches the intestine, it can undergo metabolic reduction by intestinal microflora 17, and the reductive cleavage Food dyes are added with a principal aim to give a colour to a products are rapidly absorbed 18. foodstuff, or to restore its natural colour. From the organoleptic point of view, the visual aspect is an important factor for the choice Following reductive cleavage of the azo linkage by intestinal of the products by the consumer. Thus, the synthetic food dyes bacteria, Sulfanilic acid and aminopyrazolone are produced. The occupy an important place in the class of the essential additives for pyrazolone fragment is further degraded by intestinal bacteria to food industry in the conquest of markets. Among the food dyes yield a second molecule of Sulfanilic acid 19. which are widely used is Tartrazine. It is an orange‐coloured, water soluble powder used worldwide as food additives to colour several The studies described here were performed to investigate the oral foods, drugs and cosmetics. It has the following chemical structure subchronic toxicity of Tartrazine in Wistar rats. illustrated in Fig.1. MATERIALS AND METHODS Chemicals Tartrazine (CAS 1934‐21‐0, Purity 86.7%), Sulfanilic acid (CAS 122‐ 57‐3, Purity) were purchased from Alfa Aesar (Germany), Sigma‐ Aldrich (Japan) respectively. Animals and housing Male and female Wistar rats weighing between 170 and 200 g were housed in a controlled room with a 12 h light‐dark cycle and temperature of 22 ± 2°C (animal house of the department of biology, faculty of sciences, Oujda, Morocco). They were kept in transparent polypropylene cages with free access to water and dry rat pellets feeds (Societé SONABETAIL, Oujda, Morocco). Experimental design Fig. 1: Chemical structure of Tartrazine (trisodium salt of 3­ The animals were divided into 5 groups of 6 animals each, 3 of each carboxy­5­hydroxy­ 1­(p­sulphophenyl)­4­(p sulphophenylazo) sex. All animals were treated by daily oral gavage for 90 days with a pyrazole volume of 10ml/kg. Tartrazine and Sulfanilic acid were dissolved in distilled water. Tartrazine was administered at 5, 7.5, or 10 mg/kg Moreover, this food colorant is used in cooking in many developing b.w. Sulfanilic acid was administered at 3.75 mg/kg b.w. For the countries as a substitute for saffron 1. The Acceptable Daily Intake control group it was administered with distilled water. (ADI) for humans is 0‐7.5 mg kg‐1 body weight 2. The animals were observed daily for general conditions. They were Tartrazine has been implicated as the food additive which is most weighed once every ten days during the administration period, on often responsible for allergic reactions in specific human the first and the last days of the period, and on the day of necropsy. populations 3,4,5. Daily food consumption was measured once a week. Himri et al. Int J Pharm Pharm Sci, Vol 3, Suppl 3, 2011, 159­169 At the end of week 13, all rats were deprived of food, but not water, Organ weights were obtained for the heart, lung, liver, spleen, and overnight and then blood samples were collected via the abdominal stomach. The left and right kidneys were weighed separately. aorta for hematology and serum biochemistry. Animals were then Relative organ weights were calculated based on body weights killed by exsanguination from the abdominal aorta. measured on the day of sacrifice. Hematology A portion of each organ was fixed in 10% neutral buffered formalin for histopathology, and was further processed using standard Hematological measurements and calculations were performed by method. The micro‐sections of 5 µm thicknesses were stained with using Coulter® AC·T 5diff Hematology Analyzer (Beckman Coulter Inc., hematoxylin–eosin and the prepared slides were examined under Fullerton, CA, USA). Hematological evaluations included red blood cell light microscope for histopathological. count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin Statistical analysis (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell volume distribution (RDW), blood platelet count (PLT), mean platelet Data are presented in tables or figures as the mean ± SEM. The volume (MPV), and white blood cell count (WBC), differential statistical significance of the differences between control and leukocyte percentage and reticulocyte ratio were measured. experimental groups was evaluated by Student’s t‐test using GraphPad Instat 3.06 26 For morphological examination differential counts of leukocytes were made by light microscopical observation of smear specimens RESULTS stained with a routine May– Glünwald–Giemsa protocol. Clinical observations, Body weight, Organ weights and intake Clinical biochemistry food Clinical chemistry determinations were performed by using ILab 300 Compared to the water control group, treatment with Tartrazine and (Instrumentation Laboratory Corporate Headquarters, Barcelona, Sulfanilic acid did not affect mortality, clinical signs, intake food and Spain). Parameters included Aspartate aminotransferase (AST), body weights. Body weight curves for Wistar rats during the Alanine aminotransferase (ALT), blood urea nitrogen (BUN), treatment period are shown in Fig.2. Data for the intact food and CREatinine (CRE), GLUcose (GLU), Total CHOlesterol (T‐CHO), Organ weight are shown in Table 1. Triglyceride (TG), Total Protein (TP). With regard to organ weights, statistically significant decrease of the Activity of serum ALT and AST were determined by the method of absolute right kidney weight and increases of the relative weight of Henry and al. (1960) 20. Total protein was determined according to the liver were observed in group treated with 10 mg/kg b.w of the method of Gornall and al. (1949) 21. Urea was determined in Tartrazine. Moreover, absolute lung and stomach weights were serum by the method of Tiffany and al. (1972) 22. Creatinine in significantly increased in group treated with 3.75 mg/kg b.w, of serum was estimated by the method of Fabiny and al. (1971) 23. Sulfanilic acid. Total cholesterol was estimated in serum by enzymatic colorimetric Hematological examination method according the method of Allain and al. (1974) 24. Triglycerides in serum were estimated by enzymatic colorimetric Hematology results after the treatment period did not revealed method of Trinder (1969 a) 25 a. Glucose concentration in serum was significant changes in group control and group treated with 10 estimated by enzymatic colorimetric method according to Trinder mg/kg b.w of Tartrazine. But we revealed a significantly higher (1969 b) 25 b. mean platelet volume, neutrophile and basophile; and a significantly Clinical pathology and histopathology lower blood platelet count in group treated with 7.5 mg/kg b.w of Tartrazine. In group treated with 3.75 mg/kg b.w of Sulfanilic acid At the end of the treatment, animals were exsanguinated by changes included a significantly lower blood platelet count, transecting the abdominal aorta. The body surface, the intrathracic, neutrophile, lymphocyte, monocyte, basophile, and eosinophile. intra‐abdominal organs and tissues were observed macroscopically. Hematology data are shown in Table 2. Body weight curves of wistar rats fed different concentration of Tartrazine diets for 90 days 350,00 300,00 250,00 Group control 5 mg/ (kg BW) of 200,00 Tartrazine 7.5 mg/ (kg BW) of Tartrazine 150,00 10 mg/ (kg BW) of Tartrazine 3.5 mg/ (kg BW) of 100,00 Sulfanilic acid 50,00 - - 00 10,00 20,00 30,00 40,00 50,00 60,00 70, 80,00 90,00 Fig.
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