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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
-Purity: 96.8%

Test animals

Species:
rat
Strain:
other: Crl:CD®BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston, New York
- Age at study initiation: Range from 69 days old to 80 days old depending on when they became pregnant
- Weight at study initiation: Mean of 198.1 ± 10.89g (Range 168.0 to 218.1g)
- Fasting period before study: Not specified
- Housing: in suspended, stainless steel, wire mesh cages. Nesting material was not provided because dams were scheduled to be sacrificed before expected parturition
- Diet (e.g. ad libitum): Purina® Certified Rodent Chow #5002
- Water (e.g. ad libitum): Water was from the Wilmington Suburban Water Corp.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9-23.3°C (66-74°F)
- Humidity (%): 40-60%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours (6 a.m. to 6 p.m.) of artificial illumination daily

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Methyl cellulose
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The vehicle was prepared (according to standard procedures) at least every two weeks and used as needed to prepared suspensions of the test material. Suspensions of the test material in the vehicle were prepared on the morning of each dosing day by mechanical mixture of the
test material with the vehicle followed by use of a Polytron® homogenizer until the test suspension appeared uniform. Based on a dosage volume of 10 mL/kg, nominal concentrations (pure test substance) of 0 (vehicle only), 6, 25, 100, and 400 mg/mL were prepared for the control, 60, 250, 1000, and 4000 mg/kg groups, respectively.

Two samples (10 mL each) of the test suspension for each dose level were taken at the beginning, twice during and at the end of the treatment period. For each sampling period, one sample was stored at ≤ 16°C just after mixing (fresh frozen) and the other sample was kept at room temperature for 5 hours before being stored at ≤ 16°C. Since dosing could be completed within 5 hours of preparation of the test substance suspensions, the 5-hour room temperature sample was used for comparison with the fresh frozen samples to verify stability of test substance suspensions over the time necessary for completion of dosing on each day.

On the first sampling day, an additional 10 mL sample (unscheduled) of each test substance concentration was taken just prior to dosing (as opposed to the other samples which were taken after mixing the prepared suspensions with a Polytrone homogenizer) and was fresh frozen. This extra sample was taken to see if air, put into the suspension by the homogenizer, might be affecting sampling and concentration analyses.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Each female will be mated by overnight cohabitation with a male.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Copulation will be verifed each morning by detection of a copulation plug in the vagina or on the cageboard (day 1 of gestation or Day 1G)
Duration of treatment / exposure:
10 days (Days 7-16 of gestation)
Frequency of treatment:
Daily on Days 7-16 of gestation
Duration of test:
22 days
Doses / concentrationsopen allclose all
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
4 000 mg/kg bw/day
No. of animals per sex per dose:
25 females and 25 males
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Pilot study results
- Rationale for animal assignment: Females selected for the study were ranked by their gestation day 1 body weight and assigned to control or test substance groups by random sampling from strata established within the ranked list.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs were recorded within one day of arrival, before breeding initiation, on the mornings of Days 1-22G and on the afternoons of Days 7-16G (dosing period)

BODY WEIGHT: Yes
- Time schedule for examinations: Weights were recorded within one day of arrival, before breeding initiation, and on the mornings of Days 1, 7-17, and 22G

FOOD CONSUMPTION: Yes
- Food consumption: Feeders were weighed each morning on Days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 22G to determine food consumption for intervals 1-3, 3-5, 5-7, 7-9, 9-11, 11-13, 13-15, 15-17, 17-19, and 19-22G. Food consumption for the interval was determined by subtracting the partial feeder weight (feeder with remaining food) and any spillage from the initial feeder weight (feeder filled with food).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 22
- Organs examined: Viscera, liver, uterus, ovary
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: The first live fetus and, thereafter, every other live fetus of each litter were examined
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
The litter (i.e., the proportion of affected fetuses/litter or the litter mean) was considered the experimental unit for the purpose of statistical evaluation. The level of significance selected was p≤0.05. When appropriate, the following parameters were analyzed by the indicated statistical tests:

Parameter Test for Linear Trend Pairwise Test Between Control
and Test Substance Groups

Incidence of pregnancy Cochran-Armitage Fisher's Exact
Clinical signs
Maternal death
Litters with total resorptions

Maternal body weight Orthogonal polynomial Dunnett's test when
Maternal body weight change of dose rank one-way ANOVA
Feed consumption was significant*
Liver weights (absolute & relative)

Nidations Jonckheere's Mann-Whitney U**
Live fetuses
Dead fetuses
Resorptions
Corpora lutea
Fetal weight
Percent resorptions
Incidence of fetal alterations

Mean eye measurements Mann-Whitney U

* When Bartlett's test was significant (p≤0.005), analyses were conducted on the ranks of the original values
** When more than 75% ties occurred in reproductive and fetal parameters, the Cochran-Armitage test was used to detect trends and the Fisher's exact test was applied to detect significant differences between groups.

The use of the words "significant" or "significantly" indicates a statistically significant difference between the control and test substance groups unless otherwise noted.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
For gestation days 1-6 (prior to treatment initiation), a trend was indicated for red discharge from the vaginal opening. This observation was made only for two high dose females on gestation day 1. For the remainder of gestation, no significant trends or increases in clinical signs were observed in the test substance groups (exclusive of signs associated with injuries and/or infections in animals).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females died during the course of the study. Postmortem examination revealed dosing injuries to be the cause of death. One female delivered her pups on the afternoon of gestation day 21 and was sacrificed that day. The remaining females survived until scheduled sacrifice on gestation day 22.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant trends were observed for gestation days 7-9, 15-17, and 7-17 (entire treatment interval), but no significant differences in mean body weight changes were observed when the test substance groups were compared to the control group.

For the treatment period (7-17G), the high-dose group had a slightly lower mean weight gain than that of the control group, while mean gains of the other test substance groups were comparable to that of the control group. After treatment cessation, weight gains of all the test substance groups were comparable to those of the control group.

No differences were seen in the mean maternal adjusted body weights. However, these weights and weight changes calculated with these weights for Days 1-22 and 7-22, showed significant trends. The trends were not evident when data from the high-dose group was omitted from the analysis.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significant trends were observed for gestation days 7-9, 9-11, 11-13, 15-17, and 7-17 (entire treatment interval), and mean food consumption values of the high-dose group were lower than that of the control group for all treatment period intervals (except for those of the 13-15 and 15-17G intervals, all reductions were statistically significant).

Mean food consumption values of the other test substance groups were not significantly different from the control group for any treatment period interval. After treatment cessation, mean food consumption values of all groups were comparable.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences in absolute or relative liver weights were found.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No pathological conditions which were regarded as test substance related were observed during the postmortem examination of females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were no abortions in any group.
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One animal in the high-dose had total resorptions at sacrifice, but postmortem examination revealed a puncture wound of the esophagus and white cheesy-appearing material in the thoracic cavity. As no other animals in the group had total resorptions and the resorption rate (mean number of resorptions per litter) was not increased for the high dose group, the embryo/fetal deaths for this female were attributed to the dosing injury and apparent infection.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Mean incidence of Resorptions (Standard deviation)
Group Total Early Late

Control 0.8 ± 0.28 0.8 ± 0.28 0.0 ± 0.00
60 mg/kg 0.5 ± 0.16 0.5 ± 0.16 0.0 ± 0.00
250 mg/kg 0.3 ± 0.13 0.3 ± 0.13 0.0 ± 0.00
1000 mg/kg 1.0 ± 0.29 1.0 ± 0.29 0.0 ± 0.00
4000 mg/kg 0.6 ± 0.19 0.5 ± 0.17 0.0 ± 0.05
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no dead fetuses in any group
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Group Number pregnant

Control 21/25
60 mg/kg 23/25
250 mg/kg 20/25
1000 mg/kg 21/25
4000 mg/kg 22/25

Effect levels (maternal animals)

Dose descriptor:
NOEL
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

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group Mean fetal weight (± Standard deviation)

Control 5.30 ± 0.05 g
60 mg/kg 5.43 ± 0.09 g
250 mg/kg 5.36 ± 0.11 g
1000 mg/kg 5.29 ± 0.08 g
4000 mg/kg 5.16 ± 0.07 g
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Group Mean Number of Live Fetuses (± Standard deviation)

Control 13.1 ± 0.73
60 mg/kg 13.5 ± 0.41
250 mg/kg 12.9 ± 0.80
1000 mg/kg 13.3 ± 0.61
4000 mg/kg 14.1 ± 0.33
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Group Females (± Standard deviation) Males (± Standard deviation)

Control 7.1 ± 0.65 6.0 ± 0.45
60 mg/kg 6.5 ± 0.38 7.0 ± 0.45
250 mg/kg 6.3 ± 0.67 6.6 ± 0.52
1000 mg/kg 6.1 ± 0.61 7.2 ± 0.58
4000 mg/kg 7.3 ± 0.44 6.8 ± 0.48
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no external malformations in the Control or 60, 250, or 1000 mg/kg groups. There was one external malformation in the 4000 mg/kg group. This was not was not statistically different.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no skeletal malformations in the Control or 60, 1000, or 4000 mg/kg groups. There was one skeletal malformation in the 250 mg/kg group. This was not was not statistically different.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The mean percent affected in the Control, 60, 250, 1000, and 4000 mg/kg groups (1.0, 1.2, 1.3, 0.6, and 0.6, respectively) was not statistically different.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The percentage of fetuses per litter with variations due to retarded development in the high-dose group was significantly higher than that of the control group and a significant trend was indicated for this parameter. No differences were seen in the overall incidence of variations.

Group Variations due to retarded development [Mean % affected/litter (± Standard deviation)]

Control 22.8 ± 23.04
60 mg/kg 20.3 ± 21.75
250 mg/kg 28.4 ± 26.40
1000 mg/kg 25.6 ± 20.00
4000 mg/kg 35.5 ± 21.09*

* Significantly different from control values (Dunnett's test); p≤0.05

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
fetal/pup body weight changes
other: Increased incidence of variations due to retarded development in the high-dose group (4000 mg/kg/day)

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the apparent no effect level for the dam and fetus was 1000 mg/kg/day. Thus, the test substance was not uniquely toxic to the conceptus.
Executive summary:

Groups of 25 pregnant Crl:CD®BR rats were administered by gavage 0, 60, 250, 1000, or 4000 mg/kg/day of the test substance in a 0.5% aqueous suspension of methyl cellulose on Days 7-16 of gestation.

 

Among the dams of the groups given the test substance, no compound-related mortality or clinical abnormalities were observed. For the treatment period, the high-dose group had a lower weight gain and significantly decreased food consumption compared to the control group. No other significant differences in body weight changes or food consumption were observed.

 

No pathological conditions which were regarded as compound related were observed during the postmortem examinations and no significant differences in liver weights were observed.

 

No adverse effects on reproductive parameters or significant differences in mean fetal weight were observed. However, a significant trend was indicated for mean fetal weight and the mean fetal weight of the high-dose group was lower than that of the control group. No significant differences were observed in the rates of malformations or developmental variations. The percentage of fetuses per litter with variations due to retarded development in the high-dose group was significantly higher than that of the control group and a significant trend was indicated for this parameter. No differences were seen in the overall incidence of variations.

 

Under the conditions of the study, the apparent no effect level for the dam and fetus was 1000 mg/kg/day. Thus, the test substance was not uniquely toxic to the conceptus.