Disodium 5,5'-(2-(1,3-dihydro-3-oxo-2H-indazol-2-ylidene)-1,2-dihydro-3H-indol-3-one)disulphonate

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

publication

Title:

Chronic Toxicity/Carcinogenicity study of FD & C Blue NO. 2 in Rats

Author:

J. F. BORZELLECA, G. K. HOGAN, A. KOESTNER

Year:

1985

Bibliographic source:

Fd Chem. Toxic. Vol. 23, No. 6, pp. 551-558, 1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: Long-term toxicity/carcinogenicity study.

Principles of method if other than guideline:

Long-term toxicity/carcinogenicity study of FD & C Blue NO. 2 in rats

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 5,5'-(2-(1,3-dihydro-3-oxo-2H-indazol-2-ylidene)-1,2-dihydro-3H-indol-3-one)disulphonate

EC Number:

212-728-8

EC Name:

Disodium 5,5'-(2-(1,3-dihydro-3-oxo-2H-indazol-2-ylidene)-1,2-dihydro-3H-indol-3-one)disulphonate

Cas Number:

860-22-0

Molecular formula:

C16H10N2O8S2.2Na

IUPAC Name:

disodium 3,3'-dioxo-1,1',3,3'-tetrahydro-2,2'-biindole-5,5'-disulfonate

Constituent 2

Reference substance name:

FD & C Blue NO. 2

IUPAC Name:

FD & C Blue NO. 2

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FD & C Blue NO. 2 - Molecular formula (if other than submission substance): C16H8N2O8S2.2Na- Molecular weight (if other than submission substance): 466.3572 g/mole - Substance type: Organic - Physical state: Powder - Impurities (identity and concentrations): 7% volatile matter

Test animals

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animalTEST ANIMALS- Source: Charles River Breeding Laboratories, Wilmington, MA- Age at study initiation: (P) x wks; (F1) x wks: P- 63-70 days old - Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: No data available - Fasting period before study: No data available - Housing: Animals were housed individually in stainless steel cages except during the mating, lactation and post-weaning periods of the in utero phase. Each rat was identified with a metal ear tag. If the tag was lost, the animal was re-tagged and/or toe-clipped.- Diet (e.g. ad libitum): Basal diet (Purina Rodent Chow from Ralston Purina Co., Inc., St Louis, MO), ad libitum- Water (e.g. ad libitum): No data available - Acclimation period: No data available ENVIRONMENTAL CONDITIONS- Temperature (°C): 20-21 °C - Humidity (%):40-60 % - Air changes (per hr): No data available - Photoperiod (hrs dark / hrs light): 12-hrlight/dark cycle

Administration / exposure

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: Basal diet

Details on exposure:

The F0 rats received FD & C Blue No. 2 in the diet for a minimum of 8 wk before the start of the mating period and F1 males received for 29 months; females for 30 months.

Details on mating procedure:

Details on study schedule- F1 parental animals not mated until [...] weeks after selected from the F1 litters.- Selection of parents from F1 generation when pups were [...] days of age.- Age at mating of the mated animals in the study: [...] weeks(Explain how study was performed on perents and offspring separately whatever information we have): A maximum of two rats of each sex from each litter were randomly selected for the chronic phase following completion of the in utero phase. There were 70 rats of each sex in each group at the outset of the chronic phase. Offspring were exposed to the same dietary levels of colouring as their parents. Exposure continued for approximately 29 months in males and 30 months in females. The study protocol required exposure for 30 months or to the point where survival decreased to ten rats of a sex in any group, at which point all animals of that sex were killed.- M/F ratio per cage: No data available - Length of cohabitation: No data available - Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancyNo data available - After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.: No data available - Further matings after two unsuccessful attempts: [no / yes (explain)]: No data available - After successful mating each pregnant female was caged (how): Individually - Any other deviations from standard protocol: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Assays of the homogeneity and stability of FD & C Blue No. 2 in the prepared diets were carried out before the start of the study. The concentrations of the test compound in the diets were analysed weekly during the first 13 wk of study, and monthly thereafter. In addition, all lots of the basic feed used during the study were analysed for heavy metals, chlorinated hydrocarbons and aflatoxin. These analyses demonstrated that the basic feed contained acceptably low levels of contaminants, that the diets were accurately prepared and that the dietary content of the test material was stable.

Duration of treatment / exposure:

30 months

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:0, 0.5, 1.0 and 2.0% (0, 304, 632 and 1282 mg/kg/day for male and 0, 363, 775 and 1592 mg/kg/day for female)Basis:actual ingested

No. of animals per sex per dose:

Total: 1300F0 generation:0 mg/kg bw/day: 120 male, 120 female 250 mg/kg bw/day: 60 male, 60 female500 mg/kg bw/day: 60 male, 60 female1000 mg/kg bw/day: 60 male, 60 femaleF1 generation:0 mg/kg bw/day: 140 male, 140 female 250 mg/kg bw/day: 70 male, 70 female500 mg/kg bw/day: 70 male, 70 female1000 mg/kg bw/day: 70 male, 70 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Examinations

Parental animals: Observations and examinations:

Mortality, morbidity and gross signs of toxic effects and Ophthalmoscopic examinations were examined.

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

No data available

Litter observations:

Mortality, morbidity and gross signs of toxic effects, Ophthalmoscopic examinations, haematology, clinical chemistry and urine analyses were examined.

Postmortem examinations (parental animals):

Organ weights, Gross pathology and histopathology were examined.

Postmortem examinations (offspring):

Organ weights, Gross pathology and histopathology were examined.

Statistics:

Statistical analysis were performed by using F test for the variances of the two control groups. If the variances differed, Welch's t test was used to determined equality of means, using the Smith- Satterthwaite correction for unequal variances. All tests were conducted at the 1.0% two-sided risk level. Bartlett's test was performed to determine whether groups had equal variance. The one-way ANOVA was applied using the F distribution to assess significance. If significant differences among the means were indicated, Dunnet's test was used to determine which means were significantly different from control. If a non-parametric procedure for testing equality of means was indicated, the Kruskal-Wallis test was used. If differences from control were indicated, a summed rank test was used to determine which groups differed from control. In the parametric case (equal variance) standard regression techniques were used with a test for trend and lack of fit. In the non-parametric case, Jonckheere's test for monotonic trend was used. The test for equal variance (Bartlett's test) was conducted at the 1.0% two-sided risk level. All other statistical tests were conducted at the 5.0 and 1.0% two-sided risk levels. The statistical analysis of tumour incidence data was performed using contingency table techniques. For each comparison, a 2 x 2 table was constructed from the numbers of animals with and without the event of interest in both the control and treated groups. The table was evaluated by the Fisher exact test. Tests were conducted at the 5.0 and 1.0% two-sided levels. The data on time to neoplastic lesion and the survival data were analysed for each sex separately by the series of programs included in the National Cancer Institute package for histopathologically proven tumours, time to tumour and time to death. Summaries were also prepared of total benign neoplastic lesions, total malignant neoplastic lesions and neoplastic lesion- beating animals by benign-, malignant- a

Reproductive indices:

Fertility and Pup viability were examined.

Offspring viability indices:

Yes, Pup viability were examined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

Mortality: No effect on survival of treated rats were observed as compared to control. Clinical signs: No data available Body weight: No effect on body weight of treated rats were observed as compared to control. Food consumption: Dose-related increase in food consumption was observed in treated rats as compared to control. Reproductive performance: No effect on numbers of pregnant females per group and pup viability at birth were observed in treated rats as compared to control.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 282 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effect on survival, body weight, food consumption and Reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

1 592 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effect on survival, body weight, food consumption and Reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

Mortality: No effect on pup viability at birth were observed although pup mortality were increased at 1282 mg/kg/day for male and 1592 mg/kg/day for female during lactation and weaning period and at 632 mg/kg/day for male and 1592 mg/kgday for female during weaning period as compared to control. Clinical signs: Hair loss (apparently due to friction against the cage), nasal and ocular discharge, staining of the hair in the anogenital region and soft stools were observed in treated pups but these random observations are not uncommon in the CD rat and therefore were not considered to be related to compound administration.Body weight: When treated with 1282mg/kg/day for male and 1592 mg/kg/day for female and 632 mg/kg/day for male and 1592 mg/kg/day for female, slightly decreased body weight were observed as compared to control. This was due to low number of pups prior to the random selection of pups for the F1 generation. Body weight were increased and were comparable to the control weights for most of the subsequent study period.Food consumption: Dose-related increase in food consumption was observed in treated rats as compared to control. The increased food consumption was probably due to the influence of the non-nutritive character of the test compound at these high concentrations.Test substance intake: The average consumption of the test compound by male rats was 304 mg/kg/day (0.5%), 632mg/kg/day (1.0%) and 1282 mg/kg/day (2.0%). The corresponding average intakes by female rats were 363, 775 and 1592 mg/kg/day.Organ weights:In female, When treated with 1592 mg/kg/day decrase in relative thyroid and kidney weight at 12 month and increased mean relative spleen and liver weights at 30 month were observed as compared to control. When treated with 363 mg/kg/day, decrase in relative thyroid at 12 month were observed as compared to control. Gross pathology: When treated with 1282 mg/kg/day, increase in grossly visible abnormalities in urinary bladders of treated rats were observed as compared to control. Blue discoloration of the gastro-intestinal tract was noted in the treated groups.The observed findings were consistent with those reported in the literature for aged rats of this strain.Histopathology:Non-neoplastic lesions:Mycoplasma pulmonis, infection by Corynebacterium kutcheri, Periarteritis nodosa, characterized by fibrinoid necrosis of the arterial walls,occurred most commonly in the arteries of the pancreas, mesentery and testes and atrophy of the seminiferous tubules, accompanied byoligospermia or aspermia.All non-neoplastic lesions were randomly distributed in treated and control animals and were not dose related.Neoplastic lesions:In male rats, When treated with 1282 mg/kg/day Pituitary-gland neoplasms, carcinomas/adenocarcinomas of mammary-gland and transitional-cell neoplasms of the urinary bladder were observed in treated rats as compared to control.The observed effect were not statistically significant as compared to control.Statistically significant increase in carcinomas/adenocarcinomas of mammary-gland and gliomas were observed in treated rats as compared to control. In female rats, Pituitary-gland neoplasms and malignant mammary-gland neoplasms (carcinoma, adenocarcinoma) observed in treated rats as compared to control.The observed effect were not statistically significant as compared to control.other findings:Ophthalmoscopic examinations: Focal and diffuse retinopathy, conjunctivitis, uveitis and cataracts were observed in treated rats but observed changes was not related to compound administration.Hematology: No significant effect on hematology of treated rats were observed as compared to control. Clinical chemistry: In male rats, when treated with 304, 632 and 1282 mg/kg/day decrased in fasting blood glucose levels at months 6 and 12 and at 24 month in 1282 mg/kg/day as compared to control.In female rats, When treated with 363, 775 and 1592 mg/kg/day decrased in fasting blood glucose levels at 24 month and at 6 and 12 months in 1592 mg/kg/day as compared to control.Urine-analysis: No significant effect on urine-analysis of treated rats were observed as compared to control.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 282 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effect on survival, body weight, food consumption, ophthalmoscopic examinations, hematology, clinical chemistry, urine-analysis, organ weights, gross pathology and histopathology

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 592 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effect on survival, body weight, food consumption, ophthalmoscopic examinations, hematology, clinical chemistry, urine-analysis, organ weights, gross pathology and histopathology

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Mortality summary for rats fed 0-2% FD & C Blue No. 2 in the diet for up to 30 months

		No. of deaths in group
		IA		IB		II		III		IV
	Dietary level (%)	0		0		0.5		1.0		2.0
Category of death		M	F	M	F	M	F	M	F	M	F
Unscheduled, up to month 12		1	2	1	4	2	3	1	2	3	2
Interim kill, at month 12		10	10	10	10	10	10	10	10	10	10
Unscheduled, after month 12		45	43	45	39	46	39	49	48	41	44
Terminal kill		14	15	14	17	12	18	10	10	17	13
	Total...	70	70	70	70	70	70	70	70	71*	69*

*One animal originally in the high-dose female group was found during wk 1 to be a male and was therefore transferral to the high-dose male group.

Incidence of neoplams of the urinary bladder, mammary gland and brain in male rats fed 0-2 % FD & C Blue No. 2 in the diet for up to 30 months

		No. of rats affected* in group
		IA	IB	II	III	IV
Organ and turnout	Dietary level (%)...	0	0	0.5	1.0	2.0
Urinary bladder
Transitional-cell Carcinoma		0/63	1/65	0/63	2/68	2/67
Transitional-cell Papilloma		0/63	0/65	1/63	0/68	1/67
Mammary glandɫ
Benign		1/59	3/55	1/4	1/4	0/51
Malignant		0/59	0/55	1/4	1/4	3/51
Brain
Glioma		2/70	2/70	2/70	2/70	7/71

*No. affected/no, examined.

ɫ Only grossly visible lesions or masses were examined histologically in groups II and III.

Applicant's summary and conclusion

Conclusions:

NOAEL for F0 and F1 generation was considered to be 1282 mg/kg/day for male and 1592 mg/kg/day for female rats when CD male and female rats were treated with FD & C Blue No. 2

Executive summary:

In long-term toxicity/carcinogenicity study study, CD male and female rats were treated with FD & C Blue No. 2 in the concnetration of 0, 304, 632 and 1282 mg/kg/day for male and 0, 363, 775 and 1592 mg/kg/day for female in basal diet. No effect on survival, body weight and food consumption of treated rats were observed as compared to control in F0 generation. No effect on numbers of pregnant females per group and pup viability at birth were observed in treated rats as compared to control. No effect on pup viability at birth were observed although pup mortality were increased at 1282 for male and 1592 mg/kg/day for female during lactation and weaning period and at 632 mg/kg/day for male and 1592 mg/kgday for female during weaning period as compared to control. Sufficient pups were available in F1 generation. Hair loss (apparently due to friction against the cage), nasal and ocular discharge, staining of the hair in the anogenital region and soft stools were observed in treated pups but these random observations are not uncommon in the CD rat and therefore were not considered to be related to compound administration. Slightly decreased body weight were observed at 1282 mg/kg/day for male and 1592 mg/kg/day for female and 632 mg/kg/day for male and 1592 mg/kg/day for female as compared to control. This was due to low number of pups prior to the random selection of pups for the F1 generation.Dose-related increase in food consumption was observed in treated rats as compared to control. The increased food consumption was probably due to the influence of the non-nutritive character of the test compound at these high concentrations in F1 generation. Similarly,Focal and diffuse retinopathy, conjunctivitis, uveitis and cataracts were observed in treated rats but observed changes were not related to compound administration. No significant effect on hematology, Clinical chemistry and urine-analysis of treated rats were observed as compared to control in F1 generation.At 1592 mg/kg/day decrase in relative thyroid and kidney weight at 12 month and increased mean relative spleen and liver weights at 30 month and at 363 mg/kg/day decrase in relative thyroid at 12 month in female were observed as compared to control. Blue discoloration of the gastro-intestinal tract was noted in the treated groups. In addition, Increase in grossly visible abnormalities in urinary bladders of treateed male rats at 1282 mg/kg/day were observed as compared to control in F1 ganaration. The observed findings were consistent with those reported in the literature for aged rats of this strain. Non-neoplastic mycoplasma pulmonis, infection by Corynebacterium kutcheri, Periarteritis nodosa, characterized by fibrinoid necrosis of the arterial walls, occurred most commonly in the arteries of the pancreas, mesentery and testes and atrophy of the seminiferous tubules, accompanied by oligospermia or aspermia. All non-neoplastic lesions were randomly distributed in treated and control animals and were not dose related. In male rats at 1282 mg/kg/day Neoplastic Pituitary-gland neoplasms, carcinomas/adenocarcinomas of mammary-gland and transitional-cell neoplasms of the urinary bladder were observed in treated rats as compared to control. The observed effect were not statistically significant as compared to control. Statistically significant increase in carcinomas/adenocarcinomas of mammary-gland and gliomas were observed in treated rats as compared to control. In female rats, Pituitary-gland neoplasms and malignant mammary-gland neoplasms (carcinoma, adenocarcinoma) observed in treated rats as compared to control. The observed effect were not statistically significant as compared to control. Therefore, NOAEL for F0 and F1 generation was considered to be 1282 mg/kg/day for male and 1592 mg/kg/day for female rats when CD male and female rats were treated with FD & C Blue No. 2 orally for 30 months.