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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:
key study
Study period:
12 May 2008 to 17 June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
read-across source

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Covance Research Products (CRP), Inc. (Kalamazoo, Michigan)- Age at study initiation: five to six months - Weight at study initiation: 2500-3500g - Fasting period before study: None - Housing: Animals were housed one per cage in stainless steel cages in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Cages had flattened tube grid floors and were suspended above catch pans with absorbent non-contact bedding. Cages contained a J-type feeder and a pressure activated lixit valve-type watering system. There was also a variety of stainless steel objects attached to the front of the cages for environmental enrichment. - Diet: 8 oz (~180 g) LabDiet Certified Rabbit Diet #5325 (PMI Nutrition International, St. Louis, Missouri) - Water: ad libitum- Acclimation period: 6-7 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20°C ± 1°C - Humidity (%): 40-60 - Air changes (per hr): 12-15 times/hour - Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)IN-LIFE DATES: From: 12 May 2008 To: 17 June 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
Groups of 26 time-mated rabbits will be orally gavaged 7 days/week on days 7-27 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CS-1135 solutions were prepared in a vehicle of PEG 400 and administered at a dose volume of 1 ml/kg body weight in order to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted daily based on individual body weights. Dose solutions were prepared periodically based on stability data.Dose confirmation analyses of all dose levels, plus control, were conducted on the first mix prior to administration. The homogeneity of the low-dose and the high-dose solutions was determined concurrent with dose confirmation. The method for analyzing the test material in PEG 400 was solvent dilution of dose solutions followed by analysis using gas chromatography-mass spectrometry (GC/MS) and quantitation using an internal standard method. CS-1135 was shown to be stable in PEG 400 at concentrations ranging from 2.5–500 mg/mL for at least 33 days. At a concentration of 0.25 mg/mL, CS-1135 was shown to be stable in PEG 400 for at least 14 days (Mielke, 2006). Dose solutions for the current study were prepared and used within these stability limits.
Details on mating procedure:
time-mated by supplier
Duration of treatment / exposure:
days 7-27 of gestation
Frequency of treatment:
once daily
Duration of test:
gestation days 7-27
Doses / concentrationsopen allclose all
Dose / conc.:
4 mg/kg bw/day (nominal)
Dose / conc.:
12 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
No. of animals per sex per dose:
26
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Cage side and clinical observations were conducted at least daily. Body weights were recorded on GD 0 by the supplier, daily during the dosing period, and on GD 28. Statistical analysis of body weights were performed using data collected on GD 0, 7, 10, 13, 16, 20, 24, and 28. Statistical analysis of body weight gains were conducted for the following intervals: GD 0-7, 7-10, 10-13, 13-16, 16-20, 20-24, 24-28, 7-28, and 0-28. Daily feed consumption was recorded and statistically analyzed for all animals from GD 4-28.
Ovaries and uterine content:
On GD 28, all surviving adult females (not fasted) were submitted for a complete necropsy. The liver and kidneys were weighed and organ:body weight ratios calculated. A detailed examination of the reproductive tract was performed and the number and position of implantations, viable fetuses, and resorptions were recorded. Resorptions were classified as either "early" or "late" based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form. For females with one or more viable fetuses, the number of ovarian corpora lutea were counted. The uteri of females lacking visible implantations were stained with a 10% aqueous solution of sodium sulfide and examined for evidence of early resorptions in order to verify pregnancy status. For females with one or more viable fetuses, the number of ovarian corpora lutea were counted.
Fetal examinations:
All fetuses were weighed, and given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were then euthanized by sublingual oral administration of sodium pentobarbital solution. All fetuses were also given a visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations. The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. The fetuses were sexed by examination of the gonads.Approximately one half of the fetuses in each litter were randomly selected for craniofacial examination. The heads of these fetuses were removed, placed in Bouin's fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue. All fetuses were then eviscerated, preserved in alcohol and stained with Alizarin Red S in order to visualize ossified bone. After staining, skeletons were macerated and cleared, and a thorough evaluation of the fetal skeleton was conducted.All fetal alterations will be classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development or function and/or which occurs at a relatively low incidence in the specific species/strain. The maternal necropsy and fetal examinations will be conducted such that investigators are blind to treatment group assignment.
Statistics:
Maternal BW, maternal BW gain, organ weight (absolute and relative), fetal BW and feed consumption were evaluated by Bartlett's test (a=0.01) for equality of variances. Based on the outcome of Bartlett's test, a parametric or nonparametric ANOVA was performed. If the ANOVA is significant at a=0.05, analysis by Dunnett's test (a=0.05) or the Wilcoxon Rank-Sum test (a=0.05) with Bonferroni's correction was performed, respectively. Feed consumption values were excluded from analysis if the feed is spilled or scratched. Frequency of pre- and post- implantation loss, and fetal alterations were analyzed using a censored Wilcoxon test with Bonferroni's correction. The number of corpora lutea, implantations, litter size were evaluated using a nonparametric ANOVA (a=0.05) followed by the Wilcoxon Rank-Sum test (a=0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (a=0.05) with Bonferroni's correction. Fetal sex ratios were analyzed using a binomial distribution test. Females lacking visible implantations were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method (a=0.02), and if excluded, were excluded for sound scientific reasons. Both Dunnett's test and Bonferroni's correction correct for multiple comparisons to the control group to keep the experiment-wise a=0.05. Both were reported at the experiment wise alpha level. Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results are consistent with other biological and pathological findings and historical control values.
Indices:
Calculation of Pre- and Post-Implantation Loss• Pre-implantation loss* = ((No. corpora lutea- implantations )/ No. corpora lutea) x 100• Post-implantation loss* = ((No. implantations - viable fetuses)/ No. implantations) x 100* Note: Percent pre- and post- implantation loss were determined for each litter, followed by calculation of the mean of these litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Non significantly different from controls
Ophthalmological findings:
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Deemed to be of limited toxicological significance

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Incidence not statistically significant
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes. Details on maternal toxic effects: mean body weights for all treated groups were not significantly different from controls throughout the duration of the study. However, animals in the 40 mg/kg/day dose group had a clear, treatment-related, 19.3% decrease in mean body weight gain from GD 7-28, relative to controls. This effect was largely attributable to a 79% decrease in body weight gain from GD 24-28, which correlated with decreased food consumption during this period and was partly driven by body weight losses in two animals (1906 and 1916). There were no treatment-related effects on body weight gain in the 4 or 12 mg/kg/day dose groups. In the 40 mg/kg/day dose group, feed consumption during the last week of gestation, tended to be lower than controls, with the decreases being statistically identified GD 25-28. This decrease was concomitant with body weight loss and/or decreased body weight gain during this period.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Remarks:
Maternal toxicity
Effect level:
12 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
not specified
Description (incidence and severity):
Animals in 40 mg/kg/day group had treatment-related 19.3% decrease in mean body weight from GD 7-28 relative to controls.

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
effects observed, non-treatment-related
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yesDetails on embryotoxic / teratogenic effects:There was also a treatment-related, statistically identified increase in paraovarian cysts (variation) in the fetuses of dams from this group. This finding was deemed to be of no toxicological significance due to the high prevalence in adult rabbits, the lack of any apparent parenchymal involvement, and the absence of any accompanying developmental effects. There were no treatment-related maternal or developmental effects any dose group.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOEL
Remarks:
Maternal toxicity
Effect level:
12 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
other: Maternal toxicity based on decrease body weight gain, decreased feed consumption, and increased watey contents of the cecum.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity
Remarks on result:
other: No toxicologically significant findings at the highest dose level tested
Key result
Dose descriptor:
LOEL
Remarks:
Developmental toxicity
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Developmental toxicity
Remarks on result:
other:
Remarks:
Statistically identified increase in paraovarian cysts (variation) in the fetuses of dams from the 40 mg/kg/day group

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: female reproductive system
Description (incidence and severity):
Treatment-related, statistically indentified increase in paraovarian cysts in the fetuses of dams dosed at 40 mg/kg/day. This effect was deemed to be of no toxicological significance due to high prevalence in adult rabbits, the lack of parenchymal involvement and the absence of developemental effects

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
Based on these findings the maternal no-observed-effect-level (NOEL) was considered to be 12 mg/kg/day, while the developmental toxicity no-observed-adverse-effect-level (NOAEL) was 40 mg/kg/day.