Registration Dossier
Registration Dossier
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EC number: 240-474-8 | CAS number: 16423-68-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Effects on fertility
Description of key information
The multi-generational reproductive toxicity 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.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.Preweaning offspring mortality was significantly increased at the 1.0 % and 0.5 % dose levels in the first experiment, but not in the second. However, these findings were considered to be normal variability in background mortality, because pre-weaning mortality rates were significantly higher in the control group from the second experiment when compared to the first experiment. No adverse effects on offspring growth or adult regional brain weight were observed. Thus, it was concluded that the test chemical did not adversely affect development of rats under the conditions of the experiments. A NOAEL of 1.0% test chemical in diet (corresponding to approximately 1000 mg/kg bw/d) for reprotoxic effects was derived from the study.
Link to relevant study records
- 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
- 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
- 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 - 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. - 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 - 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:
- 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: 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.
- 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
- 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
- Critical effects observed:
- no
- 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: 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
- 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
- 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
- Critical effects observed:
- no
- 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
- Behaviour (functional findings):
- not specified
- Developmental immunotoxicity:
- not specified
- 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
- 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
- Critical effects observed:
- no
- 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
- Behaviour (functional findings):
- not specified
- Developmental immunotoxicity:
- not specified
- 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
- 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
- Critical effects observed:
- no
- Reproductive effects observed:
- not specified
- 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.
Reference
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.
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 |
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 250 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Klimisch Rating 2
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Various studies have been evaluated to determine the reproductive toxicity potential of the test chemical. These include in vivo experimental studies performed on rats, mice for the test chemical. The results are mentioned below:
The multi-generational reproductive toxicity 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, 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. 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.
This result is supported by an one- generation study study where the reproductive (and psychotoxic) effects of the test chemical have been investigated in rats. Sprague-Dawley male and female rats were exposed to the test chemical administered at dietary dose levels of 0, 0.25, 0.5 and 1.0 % (corresponding to approximately 250, 500 and 1000 mg/kg/day) mixed in powdered Purina rat chow. Two separate experiments were conducted using the test chemical. The two experiments were separated by 2 years, and the second experiment was intended as a replication to resolve some of the treatment effect patterns observed in the first experiment. was fed to each treatment group for 2 weeks prior to breeding, 1-14 days during breeding (males and females housed together in pairs), then to the females during gestation and lactation, the males being discarded after confirmation of breeding (determined by the presence of sperm in the daily vaginal lavage of the females and designated as day zero of gestation). The experimental diets were freely available throughout postnatal development to the offspring of all delivering dams (up to the end of the experiments at 90-110 days of age of the offspring). Litters of fewer than eight live offspring were not retained beyond day 1 (birth was designated post natal day zero). Litters of more than 12 were reduced to 12 using a random selection procedure with sex restriction, i.e., in a way that tended to equalize the number of males and females in each litter. Body weights were measured at weekly intervals except during breeding, and food consumption was measured every third day on an arbitrarily selected subset of each treatment group (usually the first 5-15 pairs of animals enrolled in the study in each group). On the day following birth all litters were examined and data collected on litter size, sex distribution, weight, and number of dead or malformed offspring. Behavioural landmarks were evaluated according to the Cincinnati psychoteratogenicity test system for rats. The experiment was replicated after 2 years using the same exposure regimen, but without positive control in the second experiment and using different versions of the Cincinnati psychoteratogenicity test system in the two experiments. The test chemical produced no effects on paternal or offspring weight or food consumption. No significant effects of any of the measures of reproductive performance (proportion of females producing litters to those bred, the number of small litters, gestation length, litter size, sex ratio) were observed. No malformations were seen upon external examination. Preweaning offspring mortality was significantly increased at the 1.0 % and 0.5 % dose levels in the first experiment, but not in the second. However, these findings were considered to be normal variability in background mortality, because pre-weaning mortality rates were significantly higher in the control group from the second experiment when compared to the first experiment. No adverse effects on offspring growth or adult regional brain weight were observed. Thus, it was concluded that the test chemical did not adversely affect development of rats under the conditions of the experiments. A NOAEL of 1.0% test chemical in diet (corresponding to approximately 1000 mg/kg bw/d) for reprotoxic effects was derived from the study.
All of the above studies are further supported by a combined reproductive/neurobehavioral toxicity study performed to evaluate the toxic potential of the test chemical. The test chemical was administered at dietary dose levels of 0, 0.005, 0.0015 and 0.0045 % to groups of male and female Crj: CD-1 mice. The dose levels corresponded to 7.8, 22.35 and 70.43 mg/kg bw/d in males (F0 animals during premating) and 9.68, 27.86 and 82.92 mg /kg bw/d in females (F0 animals during premating) . The substance was administered during a 4-week premating period (which started at an age of 5 weeks), a 5-day mating period, and during gestation and lactation. Offspring (F1 generation) received diets until an age of 9 weeks was reached. Parental animal were weighed individually on days 0, 2, 7, 14, 21, 28 and 30 during the pre-conception period. Litter size, litter weight and sex ration of the offspring was measured at birth; offspring was weighed individually on postnatal day (PND) 0, 4, 14 and 21. Offspring was weaned at the age of 4 weeks and one male and one female were randomly selected from each litter to continue treatment until the age of 9 weeks. Animals were weighed individually at an age of 4, 5, 6, 7, 8 and 9 weeks. No significant effect were observed on survival, body weight, food consumption, compound intake of 0.045 % treated male mice and 0.015 % treated female mice as compared to control. In addition, female failed to become pregnant and abortion was observed in 0.045% dose group and increased in movement activity of exploratory behaviour at 8 weeks of age of F0 female mice as compared to control. In F1 generation, significant reduction in number of horizontal activities and on behaviour developmental parameters of male and female pups of 0.045 % dose group. In the offspring, no adverse effects on functional neurobehavioural parameters were observed. Concerning the exploratory behaviour, several parameters were changed in animal receiving the highest dose of the test chemical (significantly reduced horizontal activities and significantly increased total distance of males at week 3; increases of number of movements, movement time (statistically significant), total distance (statistically significant), average distance and average speed (statistically significant) of females at week 8). For some of the parameters (e.g. average distance in males at week 3; number of movements and average distance of females at week 8) dose-relationships could be observed. At a dose level of 0.0045 % test chemical in the diet, certain neurobehavioural parameters were statistically significantly different from control values. Therefore, NOAEL was considered to be 0.045 % (75 mg/kg/day) for male and 0.015 % (25 mg/kg/day) for female for F0 generation and 0.015 (25 mg/kg/day) for male and female mice of F1 generation when Crj: CD-1male and female mice were exposed to the test chemical orally for 35 weeks.
Based on the available results, the test chemical can be considered to be non toxic to maternal animals as well as developing fetus. Hence, the NOAEL for maternal as well as developmental toxicity can be considered to be greater than 250 mg/kg/day for the test chemical. The test chemical can be classified under the category “Not Classified” as per CLP Regulation.
Effects on developmental toxicity
Description of key information
The test chemical was evaluated for its developmental toxicity and psychotoxicity in 2 different experiments. Sprague-Dawley male and female rats were exposed to the test chemical administered at dietary dose levels of 0, 0.25, 0.5 and 1.0 % (corresponding to approximately 250, 500 and 1000 mg/kg/day) mixed in powdered Purina rat chow.
No adverse effects of the test chemical on offspring growth or adult regional brain weight were observed. Thus, it was concluded that no evidence was obtained in these two experiments to suggest that the test chemical was a developmental psychotoxin in rats at doses of up to 1.0% of the diet. Hence, the NOAEL can be considered to be 1% (1000 mg/kg/day) in diet.
Link to relevant study records
- Endpoint:
- developmental 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
- Qualifier:
- according to guideline
- Guideline:
- other: developmental and neurotoxicity test
- Principles of method if other than guideline:
- The test chemical was evaluated for for its developmental toxicity and psychotoxicity in 2 different experiments
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:Laboratory Supply Co., Indianapolis, Indiana, USA
- Age at study initiation:90-110 days of age of the offspring
- Weight at study initiation:Male (200-220 g) and female (200-220 g)
- Fasting period before study:No data available
- Housing:No data available
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:5 days - Route of administration:
- oral: feed
- Type of inhalation exposure (if applicable):
- not specified
- Vehicle:
- other: powdered Purina rat chow
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
For male : fed diets containing the dye for 2 weeks.
For female: The dye was fed to each treatment group for 2 weeks prior to breeding, 1-14 days during breeding (males and females housed together in pairs), then to the females during gestation and lactation. - Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- - Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]:cohoused
- If cohoused:
- M/F ratio per cage:1:1
- Length of cohabitation:Till the confirmation of breeding
- Further matings after two unsuccessful attempts: [no / yes (explain)]:No data
- Verification of same strain and source of both sexes: [yes / no (explain)]:No data
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:The presence of sperm in the daily vaginal lavage of the females and designated as day zero of gestation.
- Any other deviations from standard protocol:No data - Duration of treatment / exposure:
- The dye was fed to each treatment group for 2 weeks prior to breeding, 1-14 days during breeding (males and females housed together in pairs), then to the females during gestation and lactation, the males being discarded after confirmation of breeding (determined by the presence of sperm in the daily vaginal lavage of the females and designated as day zero of gestation).
- Frequency of treatment:
- Daily
- Duration of test:
- From parent to offspring generation
- Remarks:
- 0% in the diet, 0.25% , 0.5%, 1.0% in diet
- No. of animals per sex per dose:
- 19 – 22 animals/sex/dose
- Control animals:
- yes
- Details on study design:
- no data available
- Maternal examinations:
- BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
OTHER:females for reproductive success. - Ovaries and uterine content:
- No data available
- Fetal examinations:
- Other: Cincinnati Psychoteratogenicity Screening Test Battery, plus weight, food consumption, physical landmarks of development, and brain weight.
- Statistics:
- Analysis of variance for unequal group sizes, with split-plot designs where repeated measures were made, were performed on the majority of the data. Duncan a posteriori multiple range comparison tests for unequal group sizes (Kramer 1956) were used on those measures on which significant F-ratios occurred. Frequency data (e.g., mortality) were analyzed by Fisher's test for uncorrelated proportions (Guilford 1965). On preweaning tests, data on individual subjects were averaged for the entire litter or within sex and the litter was, therefore, the unit of analysis. On postweaning tests, only one male and one female was used from each litter on any given test,therefore, litter was again the unit of analysis, but no litter averaging was required. Because of the large number of tests done, and therefore, the number of statistical tests required, effects were only accepted as significant if the overall test (i.e., usually an F-test) occurred at a level ofp < 0.01 or beyond. Follow-up a posteriori individual group comparisons, however, were less stringent (p < 0.05) under the assumption that the F-test provided an adequate protection level when used at p < 0.01.
- Clinical signs:
- not specified
- Dermal irritation (if dermal study):
- not specified
- Mortality:
- not specified
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- A statistically significant treatment x days interaction was found for prebreeding and lactation body weights (both p <0.01), but not for gestation weights. Although these effects were statistically
significant they were very small (< 5%) and group comparisons revealed no significant differences between any treatment group and the control group (R3 0.0), indicating that these differences were not reliable treatment-related effects.In the second Red-3 experiment, no significant reductions were found in food consumption or body weight of the parental animals during any of the exposure periods. There were statistically significant increases found in food consumption during all three parental phases that were associated with all three dye groups.
These effects were not, however, reflected in any significant changes in body weights. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There were no significant differences found among the groups in the first experiment for food consumption prior to breeding or during gestation or lactation in the parental animals.
In the second Red-3 experiment, no significant reductions were found in food consumption or body weight of the parental animals during any of the exposure periods. There were statistically significant increases found in food consumption during all three parental phases that were associated with all three dye groups. - 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 significant reductions in day 90 brain weights were observed in either experiment. In the second experiment there was a significant treatment main-effect on cerebellar weights (p < 0.01), which, by individual group comparisons, was found to be the result of a significant increase in cerebellar weight in the R3 0.5 group (7.5%) and in the R3 0.25 group (11%) compared to the R3 0.0 control group. As before, these isolated, non-dose-dependent' increases are not thought to be treatment-related.
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- no effects observed
- Description (incidence and severity):
- No significant effects of any of the measures of reproductive performance were found in either experiment.
- Number of abortions:
- not specified
- Pre- and post-implantation loss:
- not specified
- Total litter losses by resorption:
- not specified
- Early or late resorptions:
- not specified
- Dead fetuses:
- not specified
- Changes in pregnancy duration:
- not specified
- Changes in number of pregnant:
- not specified
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- No significant reductions were found in offspring food consumption or body weight in either experiment from birth through day 90 of postnatal life. Significant increases in postweaning food consumption in the dye groups were found in both experiments (both p < 0.01), but were not reflected in altered body weights in the second experiment and were primarily in the low-dose (R3
0.25) group in the first experiment. The latter effect had disappeared by 90 days of postnatal life - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- The R3 1.0, R3 0.5, and R3 HU groups all showed significant (all p < 0.01) increases in pre-weaning mortality in the first experiment. No changes were found during later phases. The increase in
pre-weaning mortality in the dye groups was not replicated in the second experiment. In fact, the R3 1.0 group showed a significant reduction in pre-weaning mortality. It is noteworthy that pre-weaning mortality rates were significantly higher (p < 0.01) in the controls (R3 0.0) in the second experiment compared to the first. This increased level of background mortality was not due to any detectable infection in the animal colony and appears to merely represent normal variability in animal viability as they come from the supplier. - Changes in sex ratio:
- not specified
- Changes in litter size and weights:
- not specified
- Changes in postnatal survival:
- not specified
- External malformations:
- no effects observed
- Description (incidence and severity):
- No malformations were seen in the offspring based upon external examinations the day after birth
- Skeletal malformations:
- not specified
- Visceral malformations:
- not specified
- Other effects:
- no effects observed
- Description (incidence and severity):
- Behaviorally, there were no significant effects found on the tests of surface righting, pivoting, cliff avoidance, negative geotaxis, auditory startle, pre-weaning open-field, rotorod, or active avoidance in either experiment. There was no significant effect on appetitive operant discrimination learning tested only in the first experiment. There were no significant differences on tests of incisor
eruption, eye opening, testicular appearance, olfactory orientation, or water M-maze behavior tested only in the second experiment.Significant treatment effects were found in both experiments on swimming development. In the first experiment, the significant effects only occurred on the measure termed swimming-angle. On this measure both the main-effect and the treatment x days interaction were significant (both p <0.01). Individual group comparisons showed that these differences were most pronounced on day 10 and were reflected as accelerated swimming-angle development in the R3 1.0 and R3 0.25 groups. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- fetal/pup body weight changes
- changes in postnatal survival
- external malformations
- Abnormalities:
- no effects observed
- Developmental effects observed:
- not specified
- Conclusions:
- No adverse effects were found in the present experiments on the test chemical on parental weight, food consumption, or reproductive performance. The offspring were evaluated for growth and behavioral development. There was an increase in pre-weaning mortality in the high and mid-dose groups in the first experiment, but this effect was not replicated in the second experiment. No adverse effects of the test chemical on offspring growth or adult regional brain weight were observed. Thus, it was concluded that no evidence was obtained in these two experiments to suggest that the test chemical was a developmental psychotoxin in rats at doses of up to 1.0% of the diet. Hence, the NOAEL can be considered to be 1% (1000 mg/kg/day) in diet.
- Executive summary:
The test chemical was evaluated for for its developmental toxicity and psychotoxicity in 2 different experiments. Sprague-Dawley male and female rats were exposed to the test chemical administered at dietary dose levels of 0, 0.25, 0.5 and 1.0 % (corresponding to approximately 250, 500 and 1000 mg/kg/day) mixed in powdered Purina rat chow. Two separate experiments were conducted using the test chemical. The two experiments were separated by 2 years, and the second experiment was intended as a replication to resolve some of the treatment effect patterns observed in the first experiment. was fed to each treatment group for 2 weeks prior to breeding, 1-14 days during breeding (males and females housed together in pairs), then to the females during gestation and lactation, the males being discarded after confirmation of breeding (determined by the presence of sperm in the daily vaginal lavage of the females and designated as day zero of gestation). The experimental diets were freely available throughout postnatal development to the offspring of all delivering dams (up to the end of the experiments at 90-110 days of age of the offspring). Litters of fewer than eight live offspring were not retained beyond day 1 (birth was designated post natal day zero). Litters of more than 12 were reduced to 12 using a random selection procedure with sex restriction, i.e., in a way that tended to equalize the number of males and females in each litter. Body weights were measured at weekly intervals except during breeding, and food consumption was measured every third day on an arbitrarily selected subset of each treatment group (usually the first 5-15 pairs of animals enrolled in the study in each group). On the day following birth all litters were examined and data collected on litter size, sex distribution, weight, and number of dead or malformed offspring. Behavioural landmarks were evaluated according to the Cincinnati psychoteratogenicity test system for rats. The experiment was replicated after 2 years using the same exposure regimen, but without positive control in the second experiment and using different versions of the Cincinnati psychoteratogenicity test system in the two experiments.No adverse effects were found in the present experiments on the test chemical on parental weight, food consumption, or reproductive performance. The offspring were evaluated for growth and behavioral development. There was an increase in preweaning mortality in the high and mid-dose groups in the first experiment, but this effect was not replicated in the second experiment. No adverse effects of the test chemical on offspring growth or adult regional brain weight were observed. Thus, it was concluded that no evidence was obtained in these two experiments to suggest that the test chemical was a developmental psychotoxin in rats at doses of up to 1.0% of the diet. Hence, the NOAEL can be considered to be 1% (1000 mg/kg/day) in diet.
Reference
The treatment groups for Experiment 1 were test chemical as 0.0, 0.25, 0.5, or 1.0% of the diet ,and a positive control group treated with the toxin hydroxyurea on days 2-10 of life (50 mg/kg/day, s.c.); Experiment 2 was a replication of Experiment 1 with the same dose groups, but without the positive control group.Male were discarded after breeding confirmed.
Offspring mortality expressed as a % of those alive at the beginning of each phase
|
Treatment |
Days of age |
No. Of liveoffspring after culling on day 1 |
||
1-21 |
22-24 |
25-90 |
|||
Experiment 1 |
R3 1.0 |
9.3a |
0.0 |
0.7 |
161 |
R3 0.5 |
10.2a |
0.0 |
1.5 |
147 |
|
R3 0.25 |
1.5 |
0.0 |
0.0 |
128 |
|
R3 0.0 |
3.2 |
0.0 |
0.8 |
412 |
|
R3 HU |
8.6a |
0.0 |
1.5 |
219 |
|
|
|
|
|
|
|
Experiment 2 |
R3 1.0 |
9.9a |
0.0 |
6.9 |
161 |
R3 0.5 |
15.9a |
0.0 |
3.4 |
157 |
|
R3 0.25 |
23.3 |
0.0 |
1.0 |
193 |
|
R3 0.0 |
22.5 |
0.0 |
0.0 |
129 |
|
aSignificantly different from the 0% dye exposure group, p < 0.01 |
|||||
Swimming development ratings (mean _+ SE)
|
Treatment |
na |
Swimming-direction day 6 |
na |
Swimming-angle day 10 |
Experiment 1 |
R3 1.0 |
14 |
1.9±0.1 |
12 |
2.4±0.2* |
R3 0.5 |
13 |
1.6±0.1 |
13 |
2.1±0.1 |
|
R3 0.25 |
12 |
1.8±0.1 |
11 |
2.4±0.3* |
|
R3 0.0 |
35 |
1.7±0.1 |
35 |
1.8±0.1 |
|
R3 HU |
19 |
1.7±0.1 |
19 |
2.1±0.2 |
|
|
|
|
|
|
|
Experiment 2 |
R3 1.0 |
14 |
2.2±0.2** |
14 |
1.9±0.1 |
R3 0.5 |
13 |
2.6±0.2 |
13 |
2.1±0.2* |
|
R3 0.25 |
14 |
2.6±0.1 |
14 |
2.3±0.2** |
|
R3 0.0 |
11 |
2.6±0.2 |
11 |
1.9±0.2 |
|
* F-test p < 0.01, individual group comparison to R3 0.0 group, p < 0.05 ** F-test p < 0.01, individual group comparison to R3 0.0 group, p < 0.01 an represents the number of litters tested, n varies slightly due to missing data points n = 35 in the R3 0.0 group because of controls from a previous similar experiment |
|||||
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 250 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Klimisch rating 2
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Various studies have been evaluated to determine the developmental toxic effects of the test chemical. The results include in vivo experimental studies performed on rats for the test chemical. The results are mentioned below:
The test chemical was evaluated for its developmental toxicity and psychotoxicity in 2 different experiments. Sprague-Dawley male and female rats were exposed to the test chemical administered at dietary dose levels of 0, 0.25, 0.5 and 1.0 % (corresponding to approximately 250, 500 and 1000 mg/kg/day) mixed in powdered Purina rat chow. Two separate experiments were conducted using the test chemical. The two experiments were separated by 2 years, and the second experiment was intended as a replication to resolve some of the treatment effect patterns observed in the first experiment. was fed to each treatment group for 2 weeks prior to breeding, 1-14 days during breeding (males and females housed together in pairs), then to the females during gestation and lactation, the males being discarded after confirmation of breeding (determined by the presence of sperm in the daily vaginal lavage of the females and designated as day zero of gestation). The experimental diets were freely available throughout postnatal development to the offspring of all delivering dams (up to the end of the experiments at 90-110 days of age of the offspring). Litters of fewer than eight live offspring were not retained beyond day 1 (birth was designated post natal day zero). Litters of more than 12 were reduced to 12 using a random selection procedure with sex restriction, i.e., in a way that tended to equalize the number of males and females in each litter. Body weights were measured at weekly intervals except during breeding, and food consumption was measured every third day on an arbitrarily selected subset of each treatment group (usually the first 5-15 pairs of animals enrolled in the study in each group). On the day following birth all litters were examined and data collected on litter size, sex distribution, weight, and number of dead or malformed offspring. Behavioural landmarks were evaluated according to the Cincinnati psychoteratogenicity test system for rats. The experiment was replicated after 2 years using the same exposure regimen, but without positive control in the second experiment and using different versions of the Cincinnati psychoteratogenicity test system in the two experiments.No adverse effects were found in the present experiments on the test chemical on parental weight, food consumption, or reproductive performance. The offspring were evaluated for growth and behavioral development. There was an increase in preweaning mortality in the high and mid-dose groups in the first experiment, but this effect was not replicated in the second experiment. No adverse effects of the test chemical on offspring growth or adult regional brain weight were observed. Thus, it was concluded that no evidence was obtained in these two experiments to suggest that the test chemical was a developmental psychotoxin in rats at doses of up to 1.0% of the diet. Hence, the NOAEL can be considered to be 1% (1000 mg/kg/day) in diet.
This result is supported by another study where the test chemical was administered to pregnant Osborne-Mendel rats to study its teratogenic potential. Osborne-Mendel (FDA strain) female rats (12-21 wk old, 210-270g) were obtained from the FDA rat breeding colony (200 C Street, SW, Washington, DC, USA). On mating days, two females were randomly placed in cages with one male at approximately 4.30 pm. The following morning, the presence of sperm in the vaginal smear was considered to be evidence of copulation and the sperm-positive females were considered to be at day 0 of gestation. 44-47 of the females presumed to be pregnant were then placed by random number in each dose group: 0 (distilled water), 0.05, 0.1, 0.2 or 0.4%. All females were observed daily for mortality and moribundity, and observations and deviations from normality were recorded. The rats were weighed and fluid consumption was measured daily. Feed consumption was measured weekly. On day 20 of gestation, starting at 1.00 pm, the females were examined for gross abnormalities for the last time before being killed by CO2. Caesarean sections were performed and the uterus of each animal was opened and examined in situ for the presence and position of resorption sites and foetuses (dead or alive), number of corpora iutea and number of implantation sites. Four females in the 0.4% group rejected the test fluid. Their aversion to the fluid caused a progressive decrease of their body weight.Initial body weight and weight gain during the total time of gestation were similar . Significant increases in weight gain were seen during the second week for the 0.2 and 0.4% group, but they were not considered to be dose related. Mean daily feed consumption per pregnant female was significantly increased during the second week in all treated groups, but the increases during gestation (days 0-20) were not dose related. Only the 0.2% group ate significantly more feed during the entire period of gestation than did the control group. No dose-related changes were seen in maternal clinical findings, implantations, foetal viability, foetal size (weight and length) or visceral development. No dose-related teratogenesis was seen. Skeletal development was not affected; the few statistically significant increases in skeletal variations were not dose related and were considered to be random.The test chemical was neither teratogenic nor toxic to rats at dose levels of 64 to 472 mg/kg body weight/day in drinking water Hence, the NOAEL for maternal as well as developmental toxicity can be considered to be 472 mg/kg/day for the test chemical.
In a similar study, the test chemical was administered to pregnant Osborne-Mendel rats via oral gavage route to study its teratogenic potential. Osborne-Mendel (FDA strain) female rats, 13-19 weeks of age and weighing 215-270 g, were obtained from the FDA rat breeding colony (200 C Street, S.W., Washington, D.C.). On mating days, two females were randomly placed in cages with one male at approximately 4:30 pm. The following morning, the presence of sperm in the vaginal smear was considered proof of mating and the animal was considered to be at day 0 of gestation. The test chemical was administered by gavage to 40 -44 pregnant Osborne-Mendel rats at daily dose levels of 15, 30, 100, 200, 400, or 800 mg/kg on days 0-19 of gestation. Control animals were given distilled water by gavage.All females were observed daily for mortality and moribundity, and observations and deviations from normality were recorded. Animals were weighed daily. Feed consumption was measured weekly. Fluid consumption was not measured.On day 20 of gestation, starting at 1:00 pm, the females were examined for gross abnormalities for the last time before being asphyxiated by carbon dioxide. Cesarean sections were performed, and the uterus of each animal was opened and examined in situ for the presence and position of resorption sites and fetuses (dead or alive), number of corpora lutea,and number of implantation sites. Each live fetus was promptly weighed, sexed, examined for gross external malformations, and the crown-rump length was measured.Half of the fetuses were examined for skeletal variations after being fixed in alcohol, cleared, and stained with Alizarin Red S by a modification of Dawson's procedure (Dawson, 1926). The remaining fetuses were examined for internal visceral variations after being fixed in Bouin's solution and serially sectioned according to the method of Wilson. During the entire treatment period, feed consumption by the animals given 400mglkg doses was increased significantly; the increases in the animals given 30 or 800 mg/kg were of borderline significance. The only significant increase in maternal weight gain, on days 0-7 in the animals given 30 mglkg, was considered a random occurrence. No dose-related changes were seen in maternal clinical findings, implantations, fetal viability, or fetal size (weight and length). The test chemical was neither teratogenic nor toxic to rats at dose levels of up to 800 mg/kg/day given as a single dose. Hence, the NOAEL for maternal as well as developmental toxicity can be considered to be 800 mg/kg/day for the test chemical.
All of the above studies are further supported a study whose aim was to investigate the effects of the test chemical on rat fetus in the means of teratogenity and atopic diseases. 17 female Wistar albino rats were used for the study. On mating days two females were randomly mated with one male at approximately 4.30 p.m. The following morning, a vaginal smear was obtained from each female to determine whether copulation had taken place. Sperm positive dams were considered to be at day 0 of gestation. 10 pregnants served as controls and 7 served as test chemical group. In the test chemical group each animal was treated daily, from day 7 to day 11, with the dose of 5 ml of the solution which has 2 mg/ml erythrosine dissolved in distilled water by gavage. Controls were received only 5 ml distilled water in the same time period. On 20th day of gestation starting at 1 p.m., the females were sacrificed by cervical dislocation. Laparotomy was performed. The uterus was opened and examined in situ. The uterine positions of all implantation sites were noted and their condition (early or late resorption, living or dead fetuses) was determined. Each live fetus was promptly weighed and examined for congenital anomalies the crown-rump length was measured.Routine fetal histologic studies and light microscopic examination of the sections stained with H&E was applied.Mast cells are examined in the fetal dermal sections stained with Toluidine Blue. According to t-test, there was a significant difference between control and test chemical groups in the means of body weights of mothers before pregnancy (t=-2.29 SD=15 p<0.05*) and body weights of mothers after pregnancy (t=-3.46 SD=15 p<0.01**).Resorption, abortus, neural tube defects, skeletal malformation or sternebral variation were not observed in fetuses.According to t-test, there was no significant difference between control and test chemical group fetuses in the means of fetuses weights (t=-0.72 SD=15 p>0.05).The organogenesis period did not exhibit developmental retardation in the test chemical group. Therefore, it was suggested that the test chemical did not effective in organogenesis period and also did not cause any developmental retardation. The organogenesis period did not exhibit developmental retardation in the test chemical group. Therefore, it was suggested that the test chemical did not effective in organogenesis period and also did not cause any developmental retardation. There were no congenital defects, while the number of mast cells of fetal skins were increased and exocytosis was observed. This was statistically significant (U=12 p<0.01**). Hence, the NOAEL can be considered to be 2mg/ml, when Wistar albino rats were dosed with the test chemical.
Based on the available results, the test chemical can be considered to be non toxic to maternal animals as well as developing fetus. Hence, the NOAEL for maternal as well as developmental toxicity can be considered to be greater than 250 mg/kg/day for the test chemical. The test chemical can be classified under the category “Not Classified” as per CLP Regulation.
Justification for classification or non-classification
Based on the available results, the test chemical can be considered to be non toxic to maternal animals as well as developing fetus. Hence, the NOAEL for maternal as well as developmental toxicity can be considered to be greater than 250 mg/kg/day for the test chemical. The test chemical can be classified under the category “Not Classified” as per CLP Regulation.
Additional information
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