Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

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 journals

Data source

Reference

Reference Type:

publication

Title:

MULTIGENERATION STUDY OF FD & C RED NO. 3 (ERYTHROSINE) IN SPRAGUE-DAWLEY RATS

Author:

J. F. BORZELLECA and J. B. HALLAGAN

Year:

1990

Bibliographic source:

Food and Chemical Toxiology Vol. 28, No. 12, pp. 813-819, 1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 3 generation study

Principles of method if other than guideline:

Combined three generation repeated dose repro-devp. toxicity study of the test chemical in rats orally.

GLP compliance:

not specified

Limit test:

no

Justification for study design:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

EC Number:

240-474-8

EC Name:

Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Cas Number:

16423-68-0

Molecular formula:

C20H8I4O5.2Na

IUPAC Name:

disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Details on test material:

- Name of test material (as cited in study report):
D&C Red 3
- Molecular formula (if other than submission substance): C20-H6-I4-O5.2Na
- Molecular weight (if other than submission substance): 879.8424 g/mol
- Substance type: Organic
- Physical state: Solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

COBS, Charles River CD rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

EST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts, USA
- Age at study initiation: Approximately 30 days of age
- Weight at study initiation: Males: 77 to 125 g
Females: 75 to 129 g
- Fasting period before study: No data available
- Housing: The rats were housed individually in suspended wire-mesh cages except during the mating and lactation periods. During
mating period, pairs animals were housed in plastic shoe-box cages with ground corn-cob bedding. After mating and during lactation, females were individually housed in plastic shoe-box cages with ground corncob bedding and males were returned to individual cages and identified with a metal ear tag.
- Use of restrainers for preventing ingestion (if dermal): no data available
- Diet (e.g. ad libitum): Basal diet (Purina Rodent Chow), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data available

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

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

Administration / exposure

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: Basal diet (Purina Rodent Chow)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dose were prepared by blending basal diets in a twin-shell blender by appropriate addition of the test chemical before starting the study.

DIET PREPARATION

- Rate of preparation of diet (frequency): No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet (Purina Rodent Chow) were used.
- Concentration in vehicle: 0.0, 0.25, 1.0 and 4.0 %

Details on mating procedure:

- M/F ratio per cage:1:1
- Length of cohabitation:15 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:Vaginal smears were performed daily until sperm or a copulatory plug was found; this was designated as day 0 of pregnancy.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility:After the end of first mating period, female were remated with second different male from the same treatment group.
- Further matings after two unsuccessful attempts: [no / yes (explain)]:No data available
- After successful mating each pregnant female was caged (how):Each pregnant female was placed on corn-cob bedding in an individual plastic shoe-box cage and remained there until it was re-mated.
- Any other deviations from standard protocol:Each female was rested for minimum of 13 days after the pups were weaned before being mated again. Each mating was with a different male from the same treatment group. The parentage of each weanling was determined to avoid sibling mating. Litter size was reduced to 10 (5 males and 5 females if possible) on lactation day 4 by use of a random number table.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary concentrations of the compound were determined weekly by spectrophotometry during the first 13 wk of the study and monthly thereafter to determine if the diets were prepared properly and were stable. Batches were rejected if they varied from the specified concentration by c. + 10%. In addition, all batches of the basic feed were analysed for heavy metals, pesticides and aflatoxin and the following results were obtained: no pesticide residues were present at >0.05 ppm, arsenic was present at 0.26- 0.36ppm, cadmium was present at 0.12-0.19 ppm, lead was present at 0.44-0.65 ppm, mercury was present at <0.05 ppm and aflatoxin was not detected ( < 0.02 ppm).

Duration of treatment / exposure:

30 weeks

Frequency of treatment:

Daily

Details on study schedule:

-One female was placed in the cage of a male for the mating period (15 days). Vaginal smears were performed daily until sperm or a copulatory plug was found; this was designated as day 0 of pregnancy.
- Each female was rested for a minimum of 13 days after the pups were weaned before being mated again. Each mating was with a different male from the same treatment group. The parentage of each weanling was determined to avoid sibling mating.
- First generation: The F0 rats were given the appropriate control or treated diet for 69 days prior to mating. They were mated at approximately 100 days of age. The F0 rats were mated twice.
F1a: examined for external abnormalities and killed at the end of the 21-day lactation period.
F1b :F1b pups to serve as the first generation (F1) parents. The remaining F1b pups were examined for external abnormalities and killed.

- Second generation: At approximately 100 days of age, the parents were mated to produce the F2a and remated again produce F2b generation .
F2a: examined for external abnormalities and killed at the end of the 21-day lactation period.
F2b :F2b pups to serve as the second generation (F2) parents. The remaining F2b pups were examined for external abnormalities and killed.

- Third generation: At approximately 100 days of age, the parents were mated to produce the F3a and remated again produce F3b generation .
F3a: examined for external abnormalities and killed at the end of the 21-day lactation period.
F3b :F3b pups to serve as the third generation (F3) parents. The remaining F3b pups were examined for external abnormalities and killed.

The rats were observed daily for gross signs of
toxicity and mortality. Individual body weights of
parents were recorded weekly. Food consumption was recorded weekly and compound consumption was calculated in mg/kg body weight/day. Specific observations for the reproductive aspects of this study included male and female fertility, length of the gestation period, litter size, live births, and growth and survival of the pups through weaning. Parents and offspring that died during the study or were killed were examined by autopsy.

Doses / concentrations

Remarks:

Doses / Concentrations:
0.0, 0.25, 1.0 or 4.0% (0,125,500,2000mg/kg)
Basis:
no data

No. of animals per sex per dose:

Total: 200
0.0%: 25 male, 25 female
0.25%: 25 male, 25 female
1.0 %: 25 male, 25 female
4.0 %: 25 male, 25 female

Control animals:

yes, concurrent vehicle

Details on study design:

Further details on study design
- Dose selection rationale: No data available
- Rationale for animal assignment (if not random): random
- Other: No data available

Examinations

Parental animals: Observations and examinations:

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

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

BODY WEIGHT: Yes
- Time schedule for examinations: For parents:weekly
For pups: 4 and 14 days and individual pups were weight at 21 days.

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 , Food consumption was recorded weekly compound consumption was calculated in mg/kg body weight/day, except during the mating periods.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes , compound consumption was calculated in mg/kg body weight/day, except during the mating periods.

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

OTHER: No data available

Oestrous cyclicity (parental animals):

Any irregularities in the estrous cycle were investigated.

Sperm parameters (parental animals):

No data available

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no] : yes, Litter size was reduced to 10 (5 males and 5 females if possible) on lactation day 4 by use of a random number table.
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia] : The F1 pups from the first mating were examined for external abnormalities and killed at the end of the 21-day lactation period.
Body weights of pups were determined at 4 and 14 days by weighing as litters. Individual pups were weighed at 21 days.

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead] : Parents and offspring that died during the study or were killed were examined by autopsy.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:

Postmortem examinations (parental animals):

Postmortem examinations (Parent Animal)
- SACRIFICE-All rats necropsied after treatment(F0,F1,F2).
GROSS NECROPSY: Gross necropsy were performed on all parent animals.

HISTOPATHOLOGY / ORGAN WEIGHTS: Complete post-mortem examinations were conducted on any F0 generation rat that died spontaneously.
-Haematoxylin-eosin stained sections of the following tissues were prepared for 5 rats/sex/group of the F1 parent rats and histology was performed on selected tissues from 5 rats/sex/group(F2b F3b)

Postmortem examinations (offspring):

SACRIFICE: The F1a, F2a, F3a,pups killed at the end of the 21-day lactation period.
All remaining F3b, F2b, F1b offspring were killed and discarded after treatment.
GROSS NECROPSY: The F1a , F2a , F3a pups from the first mating were examined for external abnormalities . After the second mating, 25 rats/sex/group were randomly selected from the F1b pups to serve as the second generation (F1) parents. The remaining F1b pups were examined for external abnormalities and killed. Same for the F2b and F3b pups.

HISTOPATHOLOGY / ORGAN WEIGHTS:
Haematoxylin-eosin stained sections of the following tissues were prepared for 5 rats/sex/group of the (F2b ,F3b) pups.

Statistics:

All statistical analyses compared the treatment groups with the control group (significance at P < 0.05 and P < 0.01)Male and female fertility indices were compared using the chi-square test criterion with Yates' correction for 2 x 2 contingency tables and/or Fisher's Exact Test to judge the significance of differences.Gestation, 4-, 14- and 21-day survival indices were compared by the Mann-Whitney U-test to judge the significance of differences.The number of liveborn pups, parental body 30 weights (from the first week of the generation, the week prior to first mating and the last week of the generation) and organ weights were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test for equal or unequal variance using multiple comparison tables to judge the significance of differences. The combined male and female live pup body weights at lactation day 21 were compared by analysis of variance and t-test , using multiple comparison tables to judge the significance of differences.

Reproductive indices:

Male and female fertility, length of the gestation period, litter Size and live births indices were examined.
Viability indices on day 4, 14 and 21 were examined.

Offspring viability indices:

Mentioned in table below:

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The faeces of rats from all treated groups in all generations were generally coloured red. Urine from rats consuming 4.0% of the test chemical was usually red. Hair was usually coloured red or pink.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival was high for parents in all groups. One male from the 4.0% group and two females each fromthe 1.0 and 4.0% groups of the F0 generation died spontaneously; the cause of death was undetermined

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weights for the 4.0% males and females were generally decreased when compared with controls at the end of each segment. The differences from controls were occasionally statistically significant. Group mean body-weight gain during gestation among the female parents (F0) was frequently significantly reduced (P < 0.05) in the 1.0 and 4.0% groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant differences in food consumption in the treated parents

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

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

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No effect were observed on histopathology of treated rat as compared to control.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No effect were observed on histopathology of treated rat as compared to control.

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no compound-related effects on male and female fertility indices, gestation length, the number of stillborn pups, or pup survival.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

149 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

2

Key result

Dose descriptor:

NOAEL

Effect level:

255 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The faeces of rats from all treated groups in all generations were generally coloured red. Urine from rats consuming 4.0% test chemical was usually red. Hair was usually coloured red or pink.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There were no deaths among the F1 generation parents

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weights for the 4.0% males and females were generally decreased when compared with controls at the end of each segment. Group mean body-weight
gain during gestation among the female parents (F1) was frequently significantly reduced (P < 0.05) in the 1.0 and 4.0% groups

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant differences in food consumption in the treated parents

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

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 on organ weights were noted in the treated and control animals

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No compound-related effects were noted after histological examinations of selected tissues from rats from the test chemical

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No effect were observed on histopathology of treated rat as compared to control.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No effect were observed on histopathology of treated rat as compared to control.

Other effects:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no compound-related effects on male and female fertility indices, gestation length, the number of stillborn pups, or pup survival

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Effect level:

149 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

2

Key result

Dose descriptor:

NOAEL

Effect level:

255 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The faeces of rats from all treated groups in all generations were generally coloured red. Urine from rats consuming 4.0% test chemical was usually red. Hair was usually coloured red or pink.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no deaths among the F1

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weights for the 4.0% males and females were generally decreased when compared with controls at the end of each segment. Group mean body-weight gain during gestation among the female parents (F1) was frequently significantly reduced (P < 0.05) in the 1.0 and 4.0% groups

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant differences in food consumption in the treated parents

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects on organ weights were noted in the treated and control animals

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

VIABILITY (OFFSPRING):There were no deaths among the F1 and F2 pupes.

CLINICAL SIGNS (OFFSPRING):The faeces of rats from all treated groups in all generations were generally coloured red. Urine from rats consuming 4.0% FD & C Red No. 3 was usually red. Hair was usually coloured red or pink.

BODY WEIGHT (OFFSPRING):Group mean body-weight gain during gestation among the female parents (F1) was frequently significantly reduced (P < 0.05) in the 1.0 and 4.0% groups.

Body weight of pups: When treated with 4.0 %, in male and female pups significant decreased were observed in bdy weight on day 0, 4, 14 and 21 of lactation as compared to control.


SEXUAL MATURATION (OFFSPRING):No data

ORGAN WEIGHTS (OFFSPRING):No effedct were observed on organ weight of treated pups as compared to control.

GROSS PATHOLOGY (OFFSPRING):No effedct were observed on gross pathology of treated pups as compared to control.

HISTOPATHOLOGY (OFFSPRING):No effedct were observed on histopathology of treated pups as compared to control.

OTHER FINDINGS (OFFSPRING):Complete post-mortem examinations were conducted on all rat (25/sex/group) that died spontaneously.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

149 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

255 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The faeces of rats from all treated groups in all generations were generally coloured red. Urine from rats consuming 4.0% test chemical was usually red. Hair was usually coloured red or pink.In pups, hair was coloured red or pink; no other compound-related changes were noted.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no deaths among the F2-generation parents

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight for pups from the highdose (4.0%) group was consistently decreased when compared with controls at lactation days 0, 4, 14 and 21. The differences from control values were statistically significant (P < 0.05) for males and females at day 21.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant differences in food consumption

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects on organ weights were noted in these rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No compound-related effects were noted after histological examinations of selected tissues from rats from the F3b generations

Histopathological findings:

no effects observed

Description (incidence and severity):

No compound-related effects were noted after histological examinations of selected tissues from rats from the F3b generations

Other effects:

not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not specified

Effect levels (F2)

1

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

149 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

255 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Food and compound consumption for rats consuming the test chemical in a multigeneration reproduction study

Dietary concen-tration	F0
	Food intake (g/kgbody weight/day)	Compound intake (g/kg bodyweight/day)
	Male
0.0	62±4	-
0.25	62±4	156±11
1.0	65±5	654±45
4.0	76±5	3034±171
	Female
0.0	97±8	-
0.25	100±8	249±20
1.0	101±8	1007±78
4.0	118±9	4741±363
*Mean + SEM.

Food and compound consumption for rats consuming the test chemical in a multigeneration reproduction study

Dietary concen-tration	F1b
	Food intake (g/kgbody weight/day)	Compound intake (g/kg bodyweight/day)
	Male
0.0	57±3	-
0.25	60±3	149±8
1.0	60±3	602±32
4.0	75±4	3015±176
	Female
0.0	101±10	-
0.25	109±11	271±27
1.0	103±10	1033±98
4.0	122±10	4865±382
*Mean + SEM.

Summary of reproductiondata for F₁litters

 

Dietary concn	Fertility index
	Female Pregnant/ Total female mated	Index (%)	Male Fertile/ Total males mated	Index   (%)
0.0	23/25	92	23/25	92.0
0.25	23/25	92	23/25	92.0
1.0	22/23	96	22/23	95.6
4.0	23/24	96	23/24	95.8
0.0	24/25	96	24/25	96.0
0.25	23/25	92	23/25	92.0
1.0	20/23	87	20/23	87.0
4.0	24/24	100	24/24	100

Summary of reproductiondata for F₂litters

 

Dietary concn	Fertility index
	Female Pregnant/ Total female mated	Index (%)	Male Fertile/ Total males mated	Index  (%)
0.0	23/25	92.0	24/24	100.0
0.25	24/25	96.0	24/25	96.0
1.0	24/25	96.0	24/25	96.0
4.0	24/25	96.0	24/25	96.0
0.0	25/25	100.0	25/25	100.0
0.25	24/25	96.0	24/25	96.0
1.0	25/25	100.0	25/25	100.0
4.0	23/25	92.0	23/24	95.8

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 0.25% (approximately 149 and 255 mg/kg body weight/day for males and Females) respectively for F0, F1, F2 and F3 generation Sprague-dawley COBS rats dosed with the test chemical.

Executive summary:

The multi generational reproductive toxicty study was designed to examine the effects of the test chemical on Sprague Dawley rats. One-hundred male and 99 female Sprague-Dawley rats (COBS, Charles River CD rats) were used in this study. The rats were individually housed in suspended wire-mesh cages except during the mating and lactation periods. During the mating period, pairs were housed in plastic shoe-box cages with ground corn-cob bedding. After mating and during lactation, females were individually housed in plastic shoe-box cages with ground corncob bedding and males were returned to individual cages. 0.0, 0.25, 1.0 or 4.0% (0,125,500,2000mg/kg) of the test chemical mixed in basal diet was given to the test as well as control groups. One female was placed in the cage of a male for the mating period (15 days). Vaginal smears were performed daily until sperm or a copulatory plug was found; this was designated as day 0 of pregnancy. At the end of the mating period, each pregnant female was placed on corn-cob bedding in an individual plastic shoe-box cage and remained there until it was remated. Each female was rested for a minimum of 13 days after the pups were weaned before being mated again. Each mating was with a different male from the same treatment group. The parentage of each weanling was determined to avoid sibling mating. Litter size was reduced to 10 (5 males and 5 females if possible) on lactation day 4 by use of a random number table.Specific observations for the reproductive aspects of this study included male and female fertility, length of the gestation period, littersize, live births, and growth and survival of the pups through weaning. Parents and offspring that died during the study or were killed were examined byautopsy. Complete post-mortem examinations were conducted on any F 0 generation rat that died spontaneously, on all rats (25/sex/group) from the F,generation, and on 10/sex/group of the F3b pups. Haematoxylin-eosin stained sections of the following tissues were prepared for 5 rats/sex/group of the F1 parent rats and of the F3b pups: adrenal gland, colon, heart, ileum, jejunum, kidney, liver, lungs, skin, ovaries, testes and epididymis, spleen, stomach, seminal vesicles, thyroid, urinary bladder, prostate, uterus, cervix and any other tissue with a lesion noted by gross examination. Organ weights for the adrenal gland, heart, kidneys, liver, spleen, testes and thyroid were determined for the same rats.Each generation was bred twice and breeders for subsequent generations were selected after weaning of the second mating from each generation. There were no compound-related adverse effects on reproductive indices and no gross anomalies were observed. The body weights of parents and pups were significantly reduced (P < 0.05) in all generations at the 4.0% dietary concentration. Maternal body-weight gain during gestation was frequently reduced in the 1.0 and 4.0% groups. NOAEL was considered to be 0.25%  (approximately 149 and 255 mg/kg body weight/day for males and Females) respectively for F0, F1, F2 and F3 generation Sprague-dawley COBS rats dosed with the test chemical.