Reaction mass of N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo]naphthalen-2-amine and N-(2-ethylhexyl)-1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]naphthalen-1-amine

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

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 reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Repeated dose oral toxicity study of the test chemical

Author:

Borzelleca et al

Year:

1989

Bibliographic source:

Fd Chem. Toxic

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Toxicity of the test chemical was assessed in Sprague Dawley rats in life time study

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

EC Number:

247-368-0

EC Name:

Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

Cas Number:

25956-17-6

Molecular formula:

C18H16N2O8S2.2Na

IUPAC Name:

disodium 6-hydroxy-5-[(2-methoxy-3-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2-sulfonate

Details on test material:

- Name of test material: FD & C RED NO. 40 (ALLURA RED)
- IUPAC name: disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate
- Substance type: Organic
- Physical state: solid
- Purity: 88%
- Impurity: 12%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories. Inc., Wilmington, MA
- Age at study initiation: 90 days old approx.
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: rats were housed individually in stainless-steel cages except during the mating, lactation and post-weaning periods of the in utero phase
- Diet (e.g. ad libitum): Control animals received Purina Laboratory Chow and the treated animals received the appropriate dietary admixture
- Water (e.g. ad libitum): No data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21°C
- Humidity (%): 40-60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

other: Purina Laboratory Chow

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was incorporated into the basal diet using a Patterson-Kelley twin shell blender and used at dose levels of 0.0, 0.37, 1.39 and 5.19% (0, 180, 701, 2829 mg/kg bw/day males and 0, 228, 901, 3604 mg/kg bw/day for females)

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were prepared and presented weekly.
- Mixing appropriate amounts with (Type of food): Purina
Laboratory Chow
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Purina Laboratory Chow
- Concentration in vehicle: 0.0, 0.37, 1.39 and 5.19% (0, 180, 701, 2829 mg/kg bw/day males and 0, 228, 901, 3604 mg/kg bw/day for females)
- 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:

Males: 118 weeks
Females: 121 weeks

Frequency of treatment:

1 wk before mating, throughout the 3-wk breeding period, and during the gestation and lactation periods

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Males: 0.0, 0.37, 1.39 and 5.19% (0, 180, 701, 2829 mg/kg bw/day)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Females: 0.0, 0.37, 1.39 and 5.19% (0, 228, 901, 3604 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

30/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

The dietary concentrations for this study were selected based on the results of previous subchronic studies in rats

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: gross signs of toxicity, death and morbidity were recorded daily. The rats were observed twice daily (5 days/wk) during the last 6 months of the study to minimize the loss of tissue to autolysis in rats that died on test.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for wk 0 10, bi-weekly for wk 12-26, and every 4 wk thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for wk 0 10, bi-weekly for wk 12 26, and every 4 wk thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):Yes weekly for wk 0 10, bi-weekly for wk 12 26, and every 4 wk thereafter

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained from the tail vein during the study, and from the abdominal aorta at termination test were conducted at wk 13, 26, 52 and 78 and at termination
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: five rats/sex/group
- Parameters checked : haematocrit, haemoglobin, erythrocyte count, and total and differential leucocyte counts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained from the tail vein during the study, and from the abdominal aorta at termination and clinical chemistry studies were performed on at week 52 and at termination
- Animals fasted: No data
- How many animals: 5 rats/sex/group
- Parameters checked: aspartate aminotransferase, Alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, fasting glucose, total protein, sodium, potassium, chloride, carbon dioxide (termination only) and serum electrophoresis

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were obtained by housing rats overnight in individual metabolism cages. Urinary analysis was conducted at wk 13, 26, 52, 78 and at termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked : specific gravity, pH, glucose, ketones, total protein, bilirubin and sediment.

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross autopsies were conducted on all rats that died spontaneously, were killed in a moribund condition, or were killed at the end of the study. Rats were killed by exsanguination under sodium pentobarbital. Absolute and relative organ weights were determined for the heart, liver, spleen, kidneys, testes with epididymides, and thyroid and adrenal glands.


HISTOPATHOLOGY: Yes
Complete histology was conducted on all rats from the control and high-dose groups. The following tissues from these rats were examined histologically: brain, pituitary, thoracic spinal cord, eyes, oesophagus, thyroid, thymus, heart, lungs, liver, spleen, pancreas, stomach, small and large intestine, mesenteric lymph node, kidneys, adrenal, urinary bladder, uterus, prostate, ovaries, testes with epididymides, seminal vesicles, skin, rib junction, bone marrow, nerve with muscle, and any tissue masses or lesions. Histology was also conducted on animals from any group with grossly observed masses or lesions. If a potential effect was consistently noted in a tissue then that tissue was examined histologically in all rats. All excised tissues not exhausted for histology were preserved.

Statistics:

Gain in group mean body weight and total food consumption for weeks 0-54, group mean body weight at termination, and absolute and relative organ weights were analysed by the methods of Bartlett (1937), Snedecor and Cochran (1967) and Scheffe (1953). Survival data were analysed by the methods of Sachs (1959) and Snedecor and Cochran (1967). Tumour incidence data were analysed by the methods described by Thomas et al. (1977). All analyses were considered
significant at P <0.05. Time-to-tumour analyses were not performed because data correlating the time of tumour development to the time of death were not
generated.

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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no compound-related effects on survival
Localized hair loss (apparently due to friction against the cage) and nasal and ocular discharge occurred at low incidences throughout the study in control and treated rats. A red tint to the fur was noted in treated rats, and the faeces of mid- and high-dose rats were red. Palpable masses were noted with equal incidences in the control and treated groups.

Survival at the end of the study was similar in control and treated groups

BODY WEIGHT AND WEIGHT GAIN
Group mean body weights at the end of the study were decreased in rats that received FD & C Red No. 40 except for the mid-dose 701 mg/Kg bw/day males, in which they were increased.The mean body weight of the high-dose 3604 mg/Kg bw/day females at the end of the study was significantly less than that of the controls.

FOOD CONSUMPTION
Food consumption were increased in 2829 mg/Kg bw/day foe males and 3604 mg/Kg bw/day for females in the treated group but not significantly so as compared to control

HAEMATOLOGY
Few of the hematological differed significantly between control and treated rats, and none of the differences were compound related.

CLINICAL CHEMISTRY
Few of the Clinical chemistry differed significantly between control and treated rats, and none of the differences were compound related.

URINALYSIS
Few of the urinanalysis differed significantly between control and treated rats, and none of the differences were compound related.

ORGAN WEIGHTS
No compound-related changes in organ weights, were noted in rats that died on test or that were killed in a moribund state or at the end of the study.

GROSS PATHOLOGY
No compound related adverse effect observed

HISTOPATHOLOGY:
Histological evaluation revealed a variety of lesions, including neoplasm, among the control and treated rats.
The lesions were present at similar incidences in control and treated rats and appeared to be spontaneous. None of the lesions were determined to be related to the administration of FD & C Red No. 40.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

2 829 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No compound-related adverse effects were observed

2

Dose descriptor:

NOAEL

Effect level:

901 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Mean food and compound consumptions of rats in the diet in utero and throughout their lifetimes

Dietary concn (%)	Food consumption (g/kg/day)		Compound consumption (mg/kg/day)
	Males	Females	Males	Females
0.0	49.2	60.7	-	-
0.37	48.8	63.4	180	228
1.39	50.3	63.5	701	901
5.19	54.6	69.1	2829	3604

 

Table 2. Survival and group mean body weights of rats {F1} given the test chemical in the diet in utero and throughoul their lifetimes

Dietary concn of (%)	No. surviving at the end of the study ÷	Body weight (g) :
		Mean±SD	Difference from control value
Males
0.0	14	466 ±2 9	-
0.37	12	454 ± 79	-2.6
1.39	14	503 ± 81	+ 7.9
5.19	13	450 ± 51	-3.4
Females
0.0	16	399 ± 61	-
0.37	14	393 ± 75	1.5
1.39	10	368 ± 39	-7.8
5.19	15	349 ± 57*	-12.5

÷There were 50 animals m each group at the start of the study.

: At the end of the study.

The value marked with an asterisk differs significantly from the corresponding control value I P < 005t.

Table 3, Summary of neoplasms in rats (F0 that received the test chemical in the diet utero and throughout their lifetimes

Dietary concn of (%)	Males				Females
	0.0	0.37	1.39	5.19	0.0	0.37	1.39	5.19
No. of rats examined	50	37	24	50	50	49	42	50
Adrenal
Cortical carcinoma	1	0	0	0	0	0	1	2
Phaeochromocytma	1	0	1	3	0	0	0	1
Brain
Glioma	1	0	0	0	2	0	0	0
Fibrosarcoma	0	0	0	0	0	0	0	0
Haemolymphoreticular, system
Lymphocytic lymphoma	3	4	5	1	1	0	0	1
Histiocytic lymphoma	3	1	0	3	4	2	0	2
Jejunum
Adenocarcinoma	0	1	0	0	0	0	0	0
Kidney
Benign mixed tumour	1	0	0	0	1	0	0	0
Transitional cell carcinoma	1	0	0	0	0	0	0	0
Tubular cell carcinoma	0	0	1	1	0	0	0	0
Malignant mixed tumours	1	0	0	0	1	0	0	1
Hemagiosarcoma	0	0	0	1	0	0	0	0
Liver
Cholangiocarcinoma	1	0	0	0	0	0	0	0
Hepatocellular carcinoma	2	1	0	0	0	1	0	0
Neoplastic nodule	0	2	0	0	0	0	1	1
Mammary gland
Adenocarcinoma	0	1	0	0	5	5	5	3
Adenoma	0	0	0	0	0	1	0	0
Fibroadenoma	0	0	0	1	24	28	20	19
Mesenteric lymph node
Haemangioma	1	0	0	0	0	0	0	0
Ovary
Papillary cystadenoma	0	0	0	0	0	0	0	1
Scirrhous carcinoma	0	0	0	0	0	0	0	1
Pancreas
Adenoma	0	0	0	0	0	1	0	0
Carcinoma	1	0	0	2	3	0	0	2
Skin and subcutaneous tissue
Basal cell carcinoma	1	0	0	0	0	0	0	0
Spleen
Hemangioma	1	0	0	1	1	0	0	0
Hemangiosarcoma	0	1	0	0	0	1	0	0
Stomach
Squamous cell carcinoma	0	1	0	0	0	0	0	0
Fibrosarcoma	0	0	0	1	0	0	0	0
Testis
Interstitial cell adenoma	2	0	0	2	0	0	0	0
Thymus
Carcinoma	0	0	0	0	0	0	1	0
Thyroid
C-cell adenoma	1	1	0	0	0	1	0	3
Follicular carcinoma	0	0	0	1	0	0	0	0
Uterus
Adenocarcinoma	0	0	0	0	1	4	3	1
Endometrial polyp	0	0	0	0	0	3	0	2
Endometrial sternal sarcoma	0	0	0	0	3	1	1	0
Hemangiosarcoma	0	0	0	0	0	0	0	0
Squamous cell carcinoma	0	0	0	0	0	0	1	1
Sclerosing adenocarcinoma	0	0	0	0	0	1	0	0
Ear
Neurofibroma	0	0	0	0	1	0	0	0
Eye
Neurofibrosarcoma	0	1	0	0	0	0	0	0

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect level (NOAEL) for the test chemical using male and female Sprague Dawley rats in lifetime study was observed at dose concentration of 2829 and 901 mg/kg bw/day respectively

Executive summary:

Toxicity of the test chemical was assessed in Sprague Dawley rats in life time study. 30 Sprague Dawley rats per sex per dose group were fed the test chemical in diet at dose concentration of 0, 0.37, 1.39 or 5.19% (0, 180, 701, 2829 mg/kg bw/day for males and 0, 228, 901, 3604 mg/kg bw/day for females), 1 wk before mating, throughout the 3-wk breeding period, and during the gestation and lactation periods. The animals were observed for clinical signs, mortality, morbidity, body weight and food consumption changes, hematology, clinical chemistry, gross and histopathology. No significant compound related adverse effect were observed on mortality, clinical signs, body weight (except for a reduction in body weight in high-dose 3604 mg/Kg bw/day females at the end of the study), food consumption, hematology, clinical chemistry, gross and histopathology. The no observed adverse-effect level (NOAEL) in this study was 5.19% (2829 mg/kg bw/day) for male rats, and 1.39% (901 mg/kg bw/day) for female rats.