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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 August 2019 to 21 January 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled temperature area set to maintain 18°C to 24°C, in a flame-proof cabinet, capped with nitrogen
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Test substance formulations have been previously shown to be stable and homogenous over the range of concentrations used in in this study for at least 10 days when stored in a refrigerator set to maintain a target of 5°C.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The vehicle, corn oil, was dispensed approximately weekly for administration to Group 1 control animals and preparation of the test substance formulations, which were stored at room temperature (18°C to 24°C), protected from light, until use. The vehicle was stirred continuously during dosing. Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements.
- Preliminary purification step (if any): none

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 11–13 weeks old
- Weight at study initiation: 200 and 250 g on Gestation Day 0
- Fasting period before study:
- Housing: Animals were individually housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was provided ad libitum
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 26°C
- Humidity (%): 30% to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (target of 5°C), protected from light, until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was chosen as the appropriate vehicle based on the test substance’s characteristics and
the subsequent relevant OECD testing guidelines.
- Concentration in vehicle: 0, 12.5, 37.5, 150 mg/mL
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): 2IC0148 and 1IG1538
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis twice during the study period. Concentration analysis was performed on duplicate sets of all dose levels. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. Homogeneity analysis of the dose formulations was performed once during the study on duplicate sets of low and high dose groups. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration. Stability was not assessed as the test substance formulations have been previously shown to be stable and homogenous over the range of concentrations used in in this study for at least 10 days when stored in a refrigerator set to maintain a target of 5°C.
Details on mating procedure:
The females were time-mated and were received on Gestation Day 1, 2, 3, or 4.
Duration of treatment / exposure:
From gestation day 6 to gestation day (GD) 20.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control group
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
medium dose group
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
25 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The target exposure concentrations were selected based on a previous 14-day repeated-dose study and an OECD 422 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Gestation Days 0 (by supplier) and 5–21 (daily).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was quantitatively measured on Gestation Days 5–21 (daily)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Animals were subjected to a complete necropsy examination, which included evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues as well as the thyroid gland. Furthermore, kidney, liver and thyroid gland were weighed at necropsy. The thyroid gland was examined hystopathologically.

OTHER:
THYROID HORMONES EXAMINATIONS: Yes
- Time schedule for examinations: Blood samples for thyroid hormone analyses (triiodothyronine (Total T3); thyroxine (Total T4); thyroid-stimulating hormone (TSH)) were collected (prior to noon in order to avoid diurnal fluctuations in thyroid hormone levels) from a jugular vein into tubes without anticoagulants on GD 21 from all animals.
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
- Other:
- Live and dead fetuses: Yes
- Placeta examination: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: No
Statistics:
Levene’s test11 was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparison was conducted using Dunn’s test.
A Fisher’s Exact Test16 was used to conduct pairwise group comparisons of interest.
Indices:
Pre-implantation loss, post-implantation loss, sex ratio, litter % of fetuses with abnormalities

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.
Observations noted in the test substance-treated groups, primarily including thin fur cover and scabbing on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
All females were gravid, with the exception of Female No. 2520 in the 50 mg/kg bw/day group.
Mortality:
no mortality observed
Description (incidence):
All females in the control, 50, 150, and 600 mg/kg bw/day groups survived to the scheduled necropsy on Gestation Day 21.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A lower (8.1%) mean body weight gain was noted in 600 mg/kg bw/day group when the overall treatment period (Gestation Days 6–21) was evaluated. Although mean absolute body weights in this group were comparable to the control group throughout the treatment period, a statistically significantly lower corrected body weight (5.4%) and corrected body weight gain (24.2%) were noted in this group. The body weight effects noted in the 600 mg/kg bw/day group were considered test substance-related and adverse.
Mean maternal body weights, body weight gains, corrected body weights, and corrected body weight gains in the 50 and 150 mg/kg bw/day groups, and mean gravid uterine weights at all dosage levels were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean maternal food consumption, evaluated as g/animal/day, in the 50, 150, and 600 mg/kg bw/day groups was unaffected by test substance administration.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
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):
Test substance-related higher mean kidney weights (5-7%) were noted in the 50, 150, and 600 mg/kg bw/day groups; higher liver weights (4-7%) were also noted in the 150 and 600 mg/kg bw/day groups. Kidney and liver were not evaluated microscopically, but weight elevations were similar to those noted in the previous 28-day repeat dose study using the same dosage levels. There were no other test substance-related effects on organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no observations that were considered to be associated with administration of the test substance at dosage levels of 50, 150, and 600 mg/kg/day
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related histologic changes.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones: In the 150 and 600 mg/kg bw/day groups, statistically significantly higher (72.7% and 69.7%,respectively) mean TSH concentrations were noted compared to the control group. In addition, a statistically significantly lower (30.0%) mean T3 concentration was noted in the 600 mg/kg bw/day group compared to the control group. Given the lack of any effects on thyroid gland weight at any dosage level or histopathology in the 600 mg/kg bw/day group, the differences in T3 and TSH hormones were considered test substance-related but nonadverse. There were no effects on T4 concentrations at any dosage level. In the 150 mg/kg bw/day group, a statistically significantly higher (18.0%) T4 concentration was noted compared to the control group; however, the difference in T4 concentration was not observed in a dose-dependent manner.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Intrauterine survival in all dose levels was unaffected by test substance administration.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: body weight gain

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 600 mg/kg bw/day group, mean male (5.66 g), female (5.33 g), and combined (5.48 g) fetal weights were statistically significantly lower (5.39% to 5.84%) than the concurrent control group values (5.98, 5.66, and 5.81 g, respectively) and the historocal control data (5.955, 5.653, and 5.817 g, respectively). The effects on intrauterine growth at 600 mg/kg bw/day were considered test substance-related and adverse. Intrauterine growth was unaffected by test substance administration at dose levels of 50 and 150 mg/kg bw/day. The effects on fetal weight occurred together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations or developmental variations were observed in fetuses in this study.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test substance-related skeletal malformations were observed in fetuses in this study. Fetus No. 2523-03 in the 50 mg/kg bw/day group was noted with an absent lumbar vertebra. In the absence of malformations in the higher dosage levels, this malformation in a single low-dose fetus was not considered test substance-related.
Visceral malformations:
no effects observed
Description (incidence and severity):
No visceral malformations were observed in fetuses in this study.
Other effects:
no effects observed
Description (incidence and severity):
Mean absolute and relative (to the cube root of fetal body weight) anogenital distances in the 50, 150, and 600 mg/kg bw/day groups were comparable to the control group values. Differences from the control group were slight and not statistically significant.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: body weight
Description (incidence and severity):
The effects on fetal weight occurred together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects.

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

See attachments for result tables.

Applicant's summary and conclusion

Conclusions:
In the prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal and developmental toxicity was 150 mg/kg bw/day based on lower maternal body weight gain and lower fetal body weights at 600 mg/kg bw/day. The effects on fetal weight occurred together with maternal toxicity effects, but were not considered to be secondary non-specific consequence of maternal toxicity effects.