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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 August 2018 to 11 October 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
various deviations with no impact on results or integrity of the study (see Appendix 1, attached)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
various deviations with no impact on results or integrity of the study (see Appendix 1, attached)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Appearance/physical state: Clear viscous liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST SYSTEM
- Receipt: On 24 Aug 2018, time-mated female Crl:CD(SD) rats were received from Charles River Laboratories Inc., Raleigh, NC on Gestation Day 1, 2, 3, or 4. The animals were 10–12 weeks old at receipt and weighed between 200 and 248 g on Gestation Day 0.
- Justification for test system and number of animals: The Crl:CD(SD) rat is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants. The number of animals selected for this study was based on United States EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, Aug 1998 and OECD Guidelines for the Testing of Chemicals, Guideline 414, Prenatal Developmental Toxicity Study, Jan 2001, which recommend evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, this was an appropriate number to obtain a sample size of 20 females/group at termination.
- Animal identification: Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system).
- Quarantine: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to the initiation of dosing.
- Selection, assignment, replacement and disposition of animals: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Animals at extremes of body weight range were not assigned to groups. Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. After initiation of dosing, study animals were replaced with alternate animals in the event of accidental injury, non-test substance-related health issues, or similar circumstances. The disposition of all animals was documented in the Study Records.

HUSBANDRY
- Housing: Animals were individually housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal number, dosage level, and sex. Cages were arranged on the racks in group order. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.
- Environmental conditions: Target temperatures of 68 °F to 78 °F (20 °C to 26 °C) with a relative target humidity of 30 % to 70 % were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Food: PMI Nutrition International, LLC Certified Rodent LabDiet 5002 was provided ad libitum throughout the study. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Animal enrichment: Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment or to aid in maintaining the animals’ oral or gastrointestinal health.
- Veterinary care: Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
DOSE FORMULATION AND ANALYSIS
- Preparation of vehicle: The vehicle, PEG400, was dispensed weekly for administration to Group 1 control animals and adequate amounts were divided into daily aliquots, which were stored at room temperature (18 °C to 24 °C), protected from light, until use. The vehicle was stirred continuously during dosing. Details of the dispensing of the vehicle were retained in the Study Records.
- Preparation of test substance: 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 at room temperature (18 °C to 24 °C), protected from light, until use. The dosing formulations were stirred continuously during dosing. Details of the preparation and dispensing of the test substance were retained in the Study Records.
- Sample collection and analysis: Dose formulation samples were collected for analysis on 24 August 2018 (concentration: all groups; homogeneity: groups 2 and 4) and 06 September 2018 (concentration: all groups; homogeneity: not applicable). Samples to be analyzed were transferred to the Charles River Ashland Analytical Chemistry
Department.
- Analytical method: Analyses were performed by high liquid chromatography using charged aerosol detection using a validated analytical procedure.
- Concentration analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
- Homogeneity analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. 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. After acceptance of the analytical results, backup samples were discarded.
- Stability analysis: Test substance formulations have previously shown the test substance to be stable at concentrations ranging from 1 to 200 mg/mL for up to 10 days at room temperature (18 °C to 24 °C). Therefore, stability of test substance formulations was not assessed on the current study.
Details on mating procedure:
- Time-mated female Crl:CD(SD) rats were received from Charles River Laboratories Inc., Raleigh, NC on Gestation Day 1, 2, 3, or 4.
Duration of treatment / exposure:
Gestation Days 6 to 20
Frequency of treatment:
Once daily
Duration of test:
15 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
175 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Dose / conc.:
700 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
EXPERIMENTAL DESIGN
- Experimental design is summarised in Table 3 (below).
- Female No. 1660 in the 175 mg/kg/day group was removed from study on Gestation Day 7 due to body weight loss, reduced food consumption, and adverse clinical observations (dermal atonia) beginning prior to the initiation of test substance administration; this female was replaced with Female No. 1734.
- Administration of test materials: The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 6 through 20. All animals were dosed at approximately the same time each day (see Appendix 1 – Study Protocol and Deviations, attached).
- Justification of route and dose levels: The route of administration was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature. Dosage levels were selected based on the results of a previous range-finding prenatal toxicity study. In the previous study, dosage levels of 35, 175, 350, and 700 mg/kg/day were administered by oral gavage from Gestation Days 6–20 in time-mated rats. Mean body weights and food consumption were generally unaffected by test substance administration. No remarkable findings were noted at the gross examinations. Mean gravid uterine weights were slightly lower at 700 mg/kg/day, and correlating lower mean fetal weights were also noted. However, the changes were slight (< 7%), and no external malformations or variations were noted at any dosage level. Based on these results, dosage levels of 175, 350, and 700 mg/kg/day were selected for the current study.

Examinations

Maternal examinations:
IN-LIFE PROCEDURES, OBSERVATIONS AND MEASUREMENTS
- Viability: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon (see Appendix 1 – Study Protocol and Deviations, attached). Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
- Observations: The animals were removed from the cage, and a detailed clinical observation was performed once daily, beginning on the day of receipt and lasting through euthanasia. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded 1-hour postdose.
- Body weights: Animals were weighed individually on Gestation Days 0 (by supplier) and 5–21 (daily). Gravid uterine weight was collected and net body weight (the Gestation Day 21 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–21 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
- Food consumption: Food consumption was quantitatively measured on Gestation Days 5–21 (daily).
- Terminal procedures: Terminal procedures are summarized in Table 4 (below).
- Unscheduled deaths: No animals died during the course of the study.
- Scheduled euthanasia: Animals surviving until scheduled euthanasia were weighed and euthanized by carbon dioxide inhalation (including any animals that delivered).
- Necropsy: Animals were subjected to a complete necropsy examination, which included evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
- Tissue collection and preservation: Gross lesions were collected and preserved in 10% neutral buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible.
Ovaries and uterine content:
- Ovarian and uterine examination: The uterus was weighed, and the ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. The placentae were also examined.
Fetal examinations:
- Fetal examinations: Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as developmental variations or malformations. Representative photographs of all malformations, as appropriate, were included in the Study Records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus, or normal littermate, were also included in the Study Records as needed and as appropriate for comparison, when possible.
- External: Each viable fetus was examined in detail, sexed, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.
- Visceral (internal): The sex of all fetuses was confirmed by internal examination. Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The heads from these fetuses were removed and placed in Harrison’s fixative for subsequent processing and soft-tissue examination using the Wilson sectioning technique (see Appendix 1 – Study Protocol and Deviations, attached). Following examination, the carcasses and cephalic slices were discarded (see Appendix 1 – Study Protocol and Deviations, attached).
- Skeletal: The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, foetuses were stained with Alizarin Red S7 and Alcian Blue.8 The skeletal examination was made following this procedure.
Statistics:
See below
Indices:
- Intrauterine data were summarized using 2 methods of calculation as indicated below:
(1) Group mean litter basis: Postimplantation Loss/Litter = (Number dead foetuses, resorptions (Early/Late)/Group) / (Number gravid females/Group)
(2) Proportional litter loss basis: Summation per group (%) = (Sum of postimplantation loss/Litter) / (Number of litters/Group) where postimplantation loss/Litter (%) = [(Number dead foetuses, resorptions (Early/Late)/Litter) / (Number implantation sites/Litter) * 100]
- The fetal developmental findings were summarized by:
(1) Presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the Group.
(2) Considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as Summation per Group (%) = (Sum of viable fetuses affected/Litter (%)) / (Number of Litters/Group) where viable fetuses affected /Litter (%) = [(Number of viable fetuses affected/Litter) / (Number of viable foetuses/Litter) * 100]

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
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.
- Findings noted in the test substance-treated groups, including rales, red and/or clear material, and hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.
Mortality:
no mortality observed
Description (incidence):
- All females in the control, 175, 350, and 700 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 21, including Female No. 1655 in the control group that delivered on the day of scheduled necropsy (Gestation Day 21).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 175, 350, and 700 mg/kg/day groups 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):
effects observed, non-treatment-related
Description (incidence and severity):
- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 175, 350, and 700 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions.
- Statistically significantly higher mean food consumption was noted in the 175 mg/kg/day group on Gestation Day 18–19 and in the 350 mg/kg/day group on Gestation Day 8–9 (g/animal/day only), 18–19, and 6–21 (g/animal/day only); because these changes were transient, did not occur in a dose related manner, and/or were not of sufficient magnitude to affect mean body weights at these dosage levels, these findings were not considered to be test substance-related.
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:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- At the scheduled necropsy on Gestation Day 21, no test substance-related internal findings were observed at dosage levels of 175, 350, or 700 mg/kg/day.
- Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related.
- All females were determined to be gravid.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. 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:
effects observed, non-treatment-related
Description (incidence and severity):
- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
not examined
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: no effects at the highest dose level

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean male, female, and combined fetal weights (5.7 g, 5.5 g, and 5.6 g, respectively) were statistically significantly lower (8.1%, 5.2%, and 6.7%, respectively) in the 700 mg/kg/day group compared to the concurrent control group; in addition, the values were below the minimum mean values in the Charles River Ashland historical control data range (version 2018.01; 6.036 g, 5.762 g, and 5.908 g, respectively), and therefore were considered test substance-related and adverse. Mean fetal body weights in the 175 and 350 mg/kg/day groups were similar to the control group.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
- The numbers of fetuses (litters) available for morphological evaluation were 309(25), 307(25), 306(25), and 307(25) in the control, 175, 350, and 700 mg/kg/day groups, respectively.
- Malformations were observed in 0(0), 2(2), 3(3), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
- No test substance-related external malformations were noted at any dosage level. Fetus No. 1713-10 in the 350 mg/kg/day group was noted with microstomia and mandibular micrognathia. Skeletally, mandibular micrognathia consisted of mandibles that were small and fused along mandibular symphysis; both lower incisor sockets absent; tympanic rings that were fused medially. Fetus No.1701-13 in the 175 mg/kg/day group was noted with vertebral agenesis (with anury); skeletally, this finding consisted of absent vertebrae posterior to sacral vertebra No. 2, fused arches, and an absent centrum. Because the aforementioned malformations were noted in single fetuses, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the control group, they were not considered test substance-related.
- No test substance-related external developmental variations were noted. Fetus Nos. 1654-05 and 1654-06 in the 700 mg/kg/day group were noted with twinning; because this finding was a single occurrence and the mean litter proportion was not statistically significantly different from the control group, it was not considered test substance-related.
Skeletal malformations:
no effects observed
Description (incidence and severity):
- No test substance-related skeletal malformations were noted for fetuses at any dosage level. Fetus No. 1750-07 in the 350 mg/kg/day group was noted with vertebral agenesis (sacral and lumbar vertebrae and lumbar centra absent, sacral and lumbar arches unossified, and small bone precursors). The aforementioned finding was not considered test substance-related because it was noted in a single fetus and was not observed in a dose-related manner.
- No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
- No test substance-related visceral malformations were noted in fetuses at any dosage level. Visceral malformations were noted in 1 fetus each in the 175, 350, and 700 mg/kg/day groups. In the 700 mg/kg/day group, Fetus No. 1654-05 was noted with hydrocephaly (increased cavitation of both lateral and third ventricles). Situs inversus (trachea, esophagus, heart, great and major vessels, lungs, liver, stomach, pancreas, spleen, kidneys, adrenals and/or intestines were laterally transposed) was noted in Fetus Nos. 1681-10 and 1692-08 in the 175 and 350 mg/kg/day groups, respectively; Fetus No. 1692-08 was also noted with lobular agenesis of the lungs (1 lobe present, bilateral). Because the aforementioned findings were noted in single fetuses, not observed in a dose-related manner, and/or the mean litter proportions were not significantly different from the control group, they were not considered test substance-related.
- No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
- Renal papillae not fully developed (Woo and Hoar Grade 1) was noted for 3, 1, 4 and 2 fetuses in the control, 175, 350, and 700 mg/kg/day groups, respectively. Fetus No. 1721-02 in the 700 mg/kg/day group had a yellow area on the liver. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
SUMMARY OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
- The numbers of fetuses (litters) available for morphological evaluation were 309(25), 307(25), 306(25), and 307(25) in the control, 175, 350, and 700 mg/kg/day groups, respectively.
- Malformations were observed in 0(0), 2(2), 3(3), and 1(1) fetuses (litters) in the same respective groups, and were considered to be spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
350 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

ANALYSES OF DOSING FORMULATIONS

- The analyzed dosing formulations contained 85.4% to 107% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85 % to 115 %) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

- Results of the analyses of dosing formulations are summarized in Tables 6 and 7 (attached).

Applicant's summary and conclusion

Conclusions:
Based on the absence of maternal effects at any dosage level, a dosage level of 700 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when test material was administered via oral gavage to time-mated Crl:CD(SD) rats. Based on lower mean fetal weights noted at 700 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the NOAEL for embryo/fetal development.
Executive summary:

GUIDELINE

The design of the study was based on United States EPA Guideline OPPTS 870.3700 and OECD Test Guideline 414. The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

 

METHODS

Female Crl:CD(SD) rats (25 per group) were dosed via oral gavage at 0, 175, 350 or 700 mg/kg bw/day once daily during Gestation Days 6 to 20. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, gross necropsy findings, intrauterine growth and survival, and fetal morphology.

 

All females in the control, 175, 350, and 700 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 21, including 1 female in the control group that delivered on the day of scheduled necropsy (Gestation Day 21). No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

 

Mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 175, 350, and 700 mg/kg/day groups were unaffected by test substance administration.

 

RESULTS

There were no test substance-related macroscopic findings noted at any dosage level at the scheduled necropsy on Gestation Day 21. Mean male, female, and combined fetal weights in the 700 mg/kg/day group were lower (8.1%, 5.2%, and 6.7%, respectively) compared to the control group. Intrauterine growth at 175 and 350 mg/kg/day and survival at all dosage levels were unaffected by test substance administration. There were no test substance-related fetal malformations or developmental variations noted at any dosage level.

 

CONCLUSION

Based on the absence of maternal effects at any dosage level, a dosage level of 700 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when test material was administered via oral gavage to time-mated Crl:CD(SD) rats. Based on lower mean fetal weights noted at 700 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the NOAEL for embryo/fetal development.