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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 : 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
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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 Food additives are products added to the basic foodstuffs with an aim of improving its aspect, savour, taste, colour, texture, food value and conservation. Food dyes are added with a principal aim to give a colour to a foodstuff, or to restore its natural colour. From the organoleptic point of view, the visual aspect is an important factor for the choice of the products by the consumer. Thus, the synthetic food dyes occupy an important place in the class of the essential additives for food industry in the conquest of markets. Among the food dyes which are widely used is Tartrazine. It is an orange‐coloured, water soluble powder used worldwide as food additives to colour several foods, drugs and cosmetics. It has the following chemical structure illustrated in Fig.1.
The study of the carcinogenetic and mutagenetic effects of Tartrazine were established by some authors which gives variable results 6‐16. When the Tartrazine reaches the intestine, it can undergo metabolic reduction by intestinal microflora 17, and the reductive cleavage products are rapidly absorbed 18. Following reductive cleavage of the azo linkage by intestinal bacteria, Sulfanilic acid and aminopyrazolone are produced. The pyrazolone fragment is further degraded by intestinal bacteria to yield a second molecule of Sulfanilic acid 19. The studies described here were performed to investigate the oral subchronic toxicity of Tartrazine in Wistar rats. 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 carboxy5hydroxy 1(psulphophenyl)4(p sulphophenylazo) pyrazole
Moreover, this food colorant is used in cooking in many developing countries as a substitute for saffron 1. The Acceptable Daily Intake (ADI) for humans is 0‐7.5 mg kg‐1 body weight 2. Tartrazine has been implicated as the food additive which is most often responsible for allergic reactions in specific human populations 3,4,5.
The animals were divided into 5 groups of 6 animals each, 3 of each sex. All animals were treated by daily oral gavage for 90 days with a volume of 10ml/kg. Tartrazine and Sulfanilic acid were dissolved in distilled water. Tartrazine was administered at 5, 7.5, or 10 mg/kg b.w. Sulfanilic acid was administered at 3.75 mg/kg b.w. For the control group it was administered with distilled water. The animals were observed daily for general conditions. They were weighed once every ten days during the administration period, on the first and the last days of the period, and on the day of necropsy. 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, overnight and then blood samples were collected via the abdominal aorta for hematology and serum biochemistry. Animals were then killed by exsanguination from the abdominal aorta.
Organ weights were obtained for the heart, lung, liver, spleen, and stomach. The left and right kidneys were weighed separately. Relative organ weights were calculated based on body weights 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 method. The micro‐sections of 5 µm thicknesses were stained with hematoxylin–eosin and the prepared slides were examined under light microscope for histopathological.
Hematological measurements and calculations were performed by using Coulter® AC·T 5diff Hematology Analyzer (Beckman Coulter Inc., Fullerton, CA, USA). Hematological evaluations included red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell volume distribution (RDW), blood platelet count (PLT), mean platelet volume (MPV), and white blood cell count (WBC), differential leukocyte percentage and reticulocyte ratio were measured.
Statistical analysis Data are presented in tables or figures as the mean ± SEM. The statistical significance of the differences between control and 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 stained with a routine May– Glünwald–Giemsa protocol.
RESULTS Clinical observations, Body weight, Organ weights and intake food
Clinical biochemistry
Compared to the water control group, treatment with Tartrazine and Sulfanilic acid did not affect mortality, clinical signs, intake food and body weights. Body weight curves for Wistar rats during the treatment period are shown in Fig.2. Data for the intact food and Organ weight are shown in Table 1.
Clinical chemistry determinations were performed by using ILab 300 (Instrumentation Laboratory Corporate Headquarters, Barcelona, Spain). Parameters included Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), blood urea nitrogen (BUN), CREatinine (CRE), GLUcose (GLU), Total CHOlesterol (T‐CHO), Triglyceride (TG), Total Protein (TP).
With regard to organ weights, statistically significant decrease of the absolute right kidney weight and increases of the relative weight of the liver were observed in group treated with 10 mg/kg b.w of Tartrazine. Moreover, absolute lung and stomach weights were significantly increased in group treated with 3.75 mg/kg b.w, of Sulfanilic acid.
Activity of serum ALT and AST were determined by the method of Henry and al. (1960) 20. Total protein was determined according to the method of Gornall and al. (1949) 21. Urea was determined in serum by the method of Tiffany and al. (1972) 22. Creatinine in serum was estimated by the method of Fabiny and al. (1971) 23. Total cholesterol was estimated in serum by enzymatic colorimetric method according the method of Allain and al. (1974) 24. Triglycerides in serum were estimated by enzymatic colorimetric method of Trinder (1969 a) 25 a. Glucose concentration in serum was estimated by enzymatic colorimetric method according to Trinder (1969 b) 25 b.
Hematological examination Hematology results after the treatment period did not revealed significant changes in group control and group treated with 10 mg/kg b.w of Tartrazine. But we revealed a significantly higher mean platelet volume, neutrophile and basophile; and a significantly 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 changes included a significantly lower blood platelet count, neutrophile, lymphocyte, monocyte, basophile, and eosinophile. Hematology data are shown in Table 2.
Clinical pathology and histopathology At the end of the treatment, animals were exsanguinated by transecting the abdominal aorta. The body surface, the intrathracic, intra‐abdominal organs and tissues were observed macroscopically.
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 Tartrazine 7.5 mg/ (kg BW) of Tartrazine 10 mg/ (kg BW) of Tartrazine 3.5 mg/ (kg BW) of Sulfanilic acid
200,00 150,00 100,00 50,00
,0 0 90
,0 0 80
,0 0 70
,0 0
,0 0
,0 0
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50
40
30
,0 0 20
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10
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Fig. 2: Body weights of Wistar rats treated orally with different concentration of Tartrazine and Sulfanilic acid for 90 days. 160
Himri et al. Int J Pharm Pharm Sci, Vol 3, Suppl 3, 2011, 159169 Table 1: Intact food and organ weight for Wistar rats sacrificed on day 90 of subchronic feeding of Tartrazine and Sulfanilic acid
Group control
Intact food (g) Absolue (g) Liver Right kidney Left kidney Heart Lung Stomach Spleen Relative (g/100g BW) Liver Right kidney Left kidney Heart Lung Stomach Spleen
9.19 ± 0.39 7.12 ± 0.62 0.75 ± 0.04 0.76 ± 0.05 0.81 ± 0.03 1.57 ± 0.06 2.25 ± 0.12 0.55 ± 0.03 2.86 ± 0.10 0.304 ± 0.008 0.311 ± 0.009 0.33 ± 0.018 0.65 ± 0.04 0.925 ± 0.05 0.22 ± 0.012
5 mg/ (kg BW) of Tartrazine 10.18 ± 0.60 7.07 ± 0.37 0.71 ± 0.04 0.69 ± 0.05 0.86 ± 0.05 1.66 ± 0.08 2.14 ± 0.14 0.52 ± 0.12 3.03 ± 0.07 0.308 ± 0.006 0.29 ± 0.01 0.37 ± 0.01 0.72 ± 0.05 0.92 ± 0.03 0.22 ± 0.04
7.5 mg/ (kg BW) of Tartrazine 10.20 ± 0.58
10 mg/ (kg BW) of Tartrazine 9.81 ± 0.51
8.71 ± 0.71 0.77 ± 0.07 0.76 ± 0.06 0.85 ± 0.06 1.84 ± 0.1 2.35 ± 0.13 0.57 ± 0.04
8.17 ± 1.27 0.74 ± 0.08 * 0.77 ± 0.09 0.79 ± 0.06 1.74 ± 0.11 2.17 ± 0.22 0.52 ± 0.05
3.23 ± 0.15 0.28 ± 0.02 0.28 ± 0.01 0.31 ± 0.01 0.701 ± 0.06 0.88 ± 0.04 0.21 ± 0.01
2.97 ± 0.15 * 0.270 ± 0.007 0.280 ± 0.008 0.30 ± 0.01 0.67 ± 0.05 0.82 ± 0.05 0.198 ± 0.007
3.75 mg/ (kg BW) of Sulfanilic acid 10.02 ± 0.33 9.43 ± 1.13 0.84 ± 0.04 0.82 ± 0.04 0.83 ± 0.03 1.83 ± 0.09 * 2.52 ± 0.12 * 0.57 ± 0.04 3.23 ± 0.16 0.29 ± 0.009 0.291 ± 0.009 0.29 ±0.01 0.64 ± 0.03 0.89 ± 0.02 0.201 ± 0.009
Note: values represent the mean ± SEM (n=6); * p
