5-oxo-1-(4-sulphophenyl)-2-pyrazoline-3-carboxylic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

A 90 day Oral Toxicity Study Of Tartrazine, A Synthetic Food Dye, In Wistar Rats

Author:

Imane Himri, Said Bellahcen, Faiza Souna, Fatima Belmekki, Mohammed Aziz, Mohamed Bnouham, Jouhar Zoheir, Zolikha Berkia, Hassane Mekhfi, Ennouamane Saalaoui

Year:

2011

Bibliographic source:

International Journal of Pharmacy and Pharmaceutical Sciences, Vol 3, Suppl 3, 2011

Materials and methods

Principles of method if other than guideline:

13 week subchronic toxicity study was conducted to evaluate the repeated dose toxic nature of the test compound tartrazine.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate

EC Number:

217-699-5

EC Name:

Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate

Cas Number:

1934-21-0

IUPAC Name:

trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate

Constituent 2

Reference substance name:

trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo) pyrazole-3-carboxylate

IUPAC Name:

trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo) pyrazole-3-carboxylate

Details on test material:

- Name of test material (as cited in study report): Tartrazine
- Molecular formula (if other than submission substance): C16-H12-N4-O9-S2.3Na.
C16-H9-N4-O9-S2.3Na
- Molecular weight (if other than submission substance): 534.3681 g/mol
- Substance type: Organic
- Physical state: No data available
- Impurities (identity and concentrations): 86.7% pure

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: No data available
- Age at study initiation: No data available
- Weight at study initiation: 170-200 g
- Fasting period before study: No data available
- Housing: housed in transparent
polypropylene cages in a controlled room
- Diet (e.g. ad libitum): dry rat pellets feeds (Societé Sonabetail, Oujda, Morocco) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12 h light‐dark cycle

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Tartrazine was dissolved in distilled water.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): distilled water
- Concentration in vehicle: 0, 5, 7.5, or 10 mg/kg b.w.
- Amount of vehicle (if gavage): 10mL/Kg
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data available

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 5, 7.5, or 10 mg/kg b.w.
Basis:

No. of animals per sex per dose:

Total: 24
0 mg/Kg bw: 3/sex/dose
5 mg/Kg bw: 3/sex/dose
7.5 mg/Kg bw: 3/sex/dose
10 mg/Kg bw: 3/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included. General conditions

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Once every 10 days, on the first and the last days of the period, and on the day of necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, once a week
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of 13 weeks
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, all rats were deprived of food, but not water, overnight
- How many animals: All animals
- Parameters checked in table [No.?] were examined. 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.

For morphological examination differential counts of leukocytes
were made by light microscopical observation of smear specimens stained with a routine May– Glünwald–Giemsa protocol.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of 13 weeks
- Animals fasted: Yes, all rats were deprived of food, but not water, overnight
- How many animals: All animals
- Parameters checked in table [No.?] were examined. 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).

URINALYSIS: No
- Time schedule for collection of urine: No
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: No
- Dose groups that were examined: No
- Battery of functions tested: No sensory activity / grip strength / motor activity / other: No

OTHER: Organ weights were obtained for the heart, lung, liver, spleen, and stomach. Relative organ weights were calculated based on body weights measured on the day of sacrifice.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, Animals were then killed by exsanguination from the abdominal aorta. The body surface, the intrathracic, intra‐abdominal organs and tissues were observed macroscopically.

HISTOPATHOLOGY: 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.

Other examinations:

No data available

Statistics:

The statistical significance of the differences between control and experimental groups was evaluated by Student’s t‐test using GraphPad Instat 3.06

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

Clinical signs and mortality: During the administration period, no deaths occurred in animals of any group. There were no abnormal signs in all groups.

Body weight and weight gain: Compared to the water control group, treatment with Tartrazine did not affect body weights.\

Food consumption and compound intake: Compared to the water control group, treatment with Tartrazine did not affect food intake.

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination: No data available

Haematology: Hematology results did not reveal significant changes in group control and group treated with 10 mg/kg b.w of Tartrazine. But 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 was noted.

Clinical chemistry: The levels of GLU, CREA, were significantly increased in all groups compared to group control. The levels of CHOL, TG were significantly increased in group treated with 7.5, 10 mg/kg b.w of Tartrazine. There was no significant difference in the level of BUN and ALT among the different groups. The level of AST was significantly in group treated with 10 mg/kg b.w of Tartrazine. The total Protein was significantly increased in group treated with 7.5, 10 mg/kg b.w of Tartrazine.

Urinanalysis: No data available

Neurobehaviour: No data available

Organ weights: The differences in mean organ weights between control and treated groups (5, 7.5, 10 (mg/kg b.w) of Tartrazine were nonsignificant.

Gross pathology: No data available

Histopathology: All the tissue sections obtained from the stomach of experimental Wistar rats fed with 5, 7.5, 10 mg/ (kg BW) of Tartrazine. All the sections were essentially normal without any inflammatory lesion.

The histopathological examination showed lymphoid infiltrates in the Jejunum of rats fed with 7.5, 10 mg/ (kg BW) of Tartrazine.

The histopathological studies showed brown pigment deposition in the Küpffer cells and fatty degeneration of the liver in groups treated with 7.5, 10 mg/ (kg BW) of Tartrazine.

Tubular dilatation with thickened basement membrane in group treated with 5 mg/ (kg BW) of Tartrazine, tubular degeneration, and dilatation of the glomerular capillaries in group treated with 7.5 mg/ (kg BW) of Tartrazine, and intercapillary sclerosis, atrophy of glomerulus in group treated with 10 mg/ (kg BW) of Tartrazine was noted.

Effect levels

Dose descriptor:

LOAEL

Effect level:

7.5 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The Low Observed adverse effect level (LOAEL) for the test compound tartrazine is found to 7.5 mg/Kg bw.

Executive summary:

13 week subchronic toxicity study was conducted to evaluate the repeated dose toxic nature of the test compound tartrazine 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.

 

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 amino-transferase, aspartate

amino-transferase.

 

Tartrazine 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. An increase in GLU, CREA, CHOL, TG, AST, and total Protein in serum of rats treated with Tartrazine and Sulfanilic acid compared to control rats was observed 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.

 

The Low Observed adverse effect level (LOAEL) for the test compound tartrazine is found to 7.5 mg/Kg bw.