Dihydrogen (ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, aluminium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviwed publication

Data source

Reference

Reference Type:

publication

Title:

Chronic Toxicity of Food Colors,

Author:

W. H. Hansen,

Year:

1966

Bibliographic source:

Toxicology And Applied Pharmacology (1966)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Chronic repeated dose oral toxicity study was performed for test chemical to determine its toxic nature upon repeated exposure

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

EC Number:

223-339-8

EC Name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Cas Number:

3844-45-9

Molecular formula:

C37H34N2Na2O9S3

IUPAC Name:

disodium 2-({4-[ethyl(3-sulfonatobenzyl)amino]phenyl}{4-[ethyl(3-sulfonatobenzyl)iminio]cyclohexa-2,5-dien-1-ylidene}methyl)benzenesulfonate

Test material form:

not specified

Details on test material:

-IUPAC name: dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt
-commen name: Brilliant Blue FCF
- Molecular formula:C37H34N2Na2O9S3
- Molecular weight: 792.86 g/mol
- Substance type: organic
- Physical state: solid
-Smiles: C(=C1\C=C\C(=[N+](\Cc2cc(ccc2)S(=O)(=O)[O-])CC)C=C1)(/c1c(cccc1)S(=O)(=O)[O-])c1ccc(cc1)N(Cc1cc(ccc1)S(=O)(=O)[O-])CC.[Na+].[Na+]
- InChI: 1S/C37H36N2O9S3.2Na/c1-3-38(25-27-9-7-11-33(23-27)49(40,41)42)31-19-15-29(16-20-31)37(35-13-5-6-14-36(35)51(46,47)48)30-17-21-32(22-18-30)39(4-2)26-28-10-8-12-34(24-28)50(43,44)45;;/h5-24H,3-4,25-26H2,1-2H3,(H2,40,41,42,43,44,45,46,47,48);;/q;2*+1/p-2

Specific details on test material used for the study:

- Name of test material: Brilliant Blue FCF/ Blue no. 1
- Molecular formula: C37H36N2O9S3.2Na
- Molecular weight: 792.8586 g/mol
- Substance type: Organic
- Physical state: No data
- Impurities (identity and concentrations): No data

Test animals

Species:

rat

Strain:

Osborne-Mendel

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: Animals were housed individually
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

other: Feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test compound is mixed with feed at dose levels of 0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day)

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: 0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

2 years

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day)

No. of animals per sex per dose:

Total -240 male and females
0.0%(0 mg/kg bw/day) -24 male & 24 Female
0.5 %(250 mg/kg bw/day) -24 male & 24Female
1.0%(500 mg/kg bw/day) -24 male & 24 Female
2.0 %(1000 mg/kg bw/day) -24 male & 24 Female
5.0%(2500 mg/kg bw/day) -24 male & 24 Female

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the 2 yr study
- Cage side observations checked in table [No.?] were included. Mortality and abnormality

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

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- 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: No data

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: 3-, 11-, 17-, and 22-month intervals.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals
- Parameters checked in table [No.?] were examined. white blood cell count, hemoglobin, hematocrit, and differential cell count

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

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

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

OTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, Survivors were autopsied and organ weights were recorded for heart, liver, spleen, kidney, and testes. Animals that died during the experiment were autopsied, but their organ weights were not considered in the organ weight calculations.


At the end of the two-year feeding study, 225 of the 240 animals started on the experiment were examined for gross pathology.

HISTOPATHOLOGY: Yes, At the end of the two-year feeding study, 225 of the 240 animals started on the experiment were examined for microscopic pathology.

Detailed microscopic examination of lung, heart, stomach, liver, spleen small intestine, colon, bone marrow,leg bone and muscle, urinary bladder, kidney, pancreas, thyroid ( (parathyroid when present), adrenal, and prostate and testis or ovary and uterus was conducted on the formalin-fixed, haematoxylin - eosin stained paraffin embedded tissues of 21 rats from the Blue no. 1. A more limited group of tissues (kidney, liver, testis, and spleen) was examined from 43 rats.

Other examinations:

No data

Statistics:

No data

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

No effect was observed on mortality of the animals at dietary levels up to 5% (2500 mg/Kg bw/day).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant growth inhibition occurred with the animals fed test chemical

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No effect was observed on of animals at dietary levels up to 5% (2500 mg/Kg bw/day).

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects was observed on weights of heart, liver, kidney, spleen, and testis of the animals that received Blue No. 1 at dietary levels up to 5% (2500 mg/Kg bw/day).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related change in gross pathology. The usual gross pathologic conditions of old rats were seen in all groups, with no significant difference in distribution. The most common lesions were tumors, pneumonia (mostly chronic) of varying degrees? chronic nephritis of the usual type seen in our older rats, and testicular atrophy.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related change in microscopical examination. The most common tumors were mammary adenocarcinomas, fibroadenomas, and pulmonary lymphosarcomas.

In addition to the above mentioned common tumors, the Blue No. 1 animals showed the following tumors: at the 2500 mg/Kg bw/day level, one animal each had a hepatoma, a papillary colloid adenoma of the thyroid, and a carcinoma of the prostate; one animal listed with a mammary adenocarcinoma also had a subcutaneous fibroma. At the 1000 mg/kg bw/day level, one animal each had an adenocarcinoma of the kidney, a liposarcoma of the posterior mediastinum, and an extensive lymphoblastoma of the abdominal serosa and lymph nodes. At the 500 mg/kg bw/day level, one animal each had an adrenal cortical adenoma and a subcutaneous malignant angioma. At the 250 mg/Kg bw/day level, one animal each had a subcutaneous lipoma and a mammary carcinoma. In the control group, one animal had a cecal adenoma. The common incidental lesions were pneumonia (mostly chronic) of varying degrees and chronic nephritis of the usual type seen in our older rats There was a moderate degree of testicular atrophy in one of the rats in the 1000 mg/Kg bw/day group, and there was marked atrophy in one of the controls; remaining sections of testis showed no more than slight atrophy. Other major incidental lesions included an ulcerated inflammatory mass in the cecum of a rat on the 2500 mg/Kg bw/day diet, and three large, chronic, tubo-ovarian abscesses( one each on the 1000 and 250 mg/Kg bw/day diets, and control groups)

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

No data

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant tretament related alterations were noted at the mentioned dose level

Remarks on result:

other: No toxic effects were observed

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table: Incidence of major tumors in rats fed test chemical

Tumor	Dietary level
	Controls	250	500	1000	2500
Mammary adenocarcinoma	2	2	2	-	1
Mammary fibroadenoma 3 2 3 3 1 2 5 2 1 4	3	3	1	5	1
Pulmonary lymphosarcoma	1	2	1	-	1
Endometrial sarcoma	-	1	-	-	-
Rats with tumors	7	10	7	9	6

 

Applicant's summary and conclusion

Conclusions:

Based on all the available data, it was concluded that the No observed Adverse Effect level (NOAEL) for test chemical was considered to be 2500 mg/kg bw/day in Osborne-Mendel male and female rats for 2 years feeding study.

Executive summary:

Chronic repeated dose oral toxicity study was performed for test chemical to determine its toxic nature upon repeated exposure. The test chemical was mixed with feed at dose levels of0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day) and fed for 2 years to 24 male and 24 female/dose Osborne Mendel weanling rats. During the study period, the animals were observed for mortality, any abnormality, body weight changes, hematology and were subjected to gross and microscopic pathology. No mortality and abnormality was noted during the study period. No significant growth inhibition occurred with the animals fed with test chemical . Also, no effects was observed on hemotological parameters and weights of heart, liver, kidney, spleen, and testis of the animals that received test chemical at dietary levels up to 5%(2500 mg/Kg bw/day). There was no treatment-related change in gross pathology. The usual gross pathologic conditions of old rats were seen in all groups, with no significant difference in distribution. The most common lesions were tumors, pneumonia (mostly chronic) of varying degrees? chronic nephritis of the usual type seen in our older rats, and testicular atrophy. There was no treatment-related change in microscopical examination. The most common tumors were mammary adenocarcinomas, fibroadenomas, and pulmonary lymphosarcomas. Based on the above considerations, the No observed Adverse Effect level (NOAEL) for test chemical was considered to be 2500 mg/kg bw/day in Osborne-Mendel male and female rats for 2 years feeding study.