Registration Dossier

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:
24 July 2014 to 19 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
various with no impact on the result of the study (see Appendix A, Attached)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
various with no impact on the result of the study (see Appendix A, Attached)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

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

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION
- Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. WIL Research has historical control 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 the study was based on the United States EPA Health Effects Test Guidelines: OPPTS 870.3700, Prenatal Development Toxicity Study, Aug-1998 and the OECD Guidelines for the Testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, 22-Jan-2001, which recommends evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, 25 females/group was an appropriate number of animals to obtain a sample size of 20 at termination.
- Crl:CD(SD) rats (125 females) were received in good health from Charles River Laboratories, Inc., Stone Ridge, NY, on 24-Jul-2014. The animals were approximately 80 days old upon receipt. Each female was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each rat was uniquely identified by a Monel metal ear tag displaying the animal number and housed for a minimum of 12 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behavior.

ANIMAL HOUSING
- Upon arrival and until pairing, all rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week.
- The rats were paired for mating in the home cage of the male.
- Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages; nesting material was not required as the females were euthanized prior to the date of expected parturition.
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National
Research Council, 2011). The animal facilities at WIL Research are fully accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records. The feeders were changed and sanitized once per week.
- Municipal water supplying the facility was sampled for contaminants according to WIL Research SOPs. The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.
- Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet
were provided ad libitum throughout the acclimation period and during the study.


ENVIRONMENTAL CONDITIONS
- All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71 °F ± 5 °F (22 °C ± 3 °C) and 50 % ± 20 %, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 69.8 °F to 70.7°F (21.0 °C to 21.5 °C) and mean daily relative humidity ranged from 47.2 % to 58.6 % during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Remarks:
Lot number 1DB0384; expiry date 31 January 2015
Details on exposure:
PREPARATION
- The vehicle suspension was prepared approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light.
- The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.
- Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature , protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

ORGANIZATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon-shafted, stainless steel ball-tipped dosing cannula once daily during gestation days 6-19.
- The dosage volume for all groups was 5 mL/kg.
- Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose.
- All animals were dosed at approximately the same time each day.
- Study group assignment is shown in the table below.
- Dosage levels were selected based on results of a previous 28-day toxicity study in rats (Haas, 2015, WIL-168211) in which dosage levels of 250, 500, and 1000 mg/kg/day were used. There were no apparent effects on body weight gains or food consumption in the previous study and no significant clinical signs noted in either sex. A single female in the 1000 mg/kg/day group was found dead on study day 18; however, no cause of death could be determined for this animal. Based on the limited toxicity noted in the previous study, dosage levels of 250, 500, and 1000 (limit dose for OECD 414 studies) mg/kg/day were chosen for evaluation on the current study.
- The selected route of administration for this study 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSES
- Homogeneity and stability of the test substance in the vehicle for up to 7 days were previously demonstrated at concentrations spanning the range used in the current study (Haas, 2015, WIL-168211).
- Samples for concentration and/or homogeneity determinations were collected from the middle stratum of the first and last control group dosing formulations and from the top, middle, and bottom strata of the first and last test substance dosing formulations.
- Samples were shipped at room temperature, protected from light, to Wildlife International for analysis. All analyses were conducted by Wildlife International using a validated method (Wildlife International Project No. 796C-102).
Details on mating procedure:
ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS AND BREEDING PROCEDURES
- At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding.
- Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females.
- A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained.
- The selected females were approximately 13 weeks old when paired for breeding.
- Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily.
- The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.
- The experimental design consisted of 3 test substance-treated groups and 1 control group, composed of 25 rats per group.
- The bred females were assigned to groups using a WTDMS computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design.
- Animals not assigned to the study were transferred to the WIL Research stock colony or euthanized by carbon dioxide inhalation and discarded.
- Body weight values ranged from 216 g to 302 g on gestation day 0.
Duration of treatment / exposure:
Gestation days 6 to 19
Frequency of treatment:
Daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 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:
- See diagram summarising study design (attached).

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period).
- Animals were also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
- Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily).
- Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20.
- Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 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
- Individual food consumption was recorded on gestation days 0 and 6-20 (daily).
- Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data.
- The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.
Ovaries and uterine content:
GESTATION DAY 20 LAPAROHYSTERECTOMY
- Laparohysterectomies and macroscopic examinations were performed blind to treatment group.
- All females were euthanized on gestation day 20 by carbon dioxide inhalation.
- The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised.
- The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined.
- The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.
Fetal examinations:
FETAL MORPHOLOGICAL EXAMINATION
- Fetal examinations were performed blind to treatment group.
- Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number.
- The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded.
- Nonviable fetuses (if the degree of autolysis was minimal or absent) were examined, the crown-rump length measured, weighed, sexed, and tagged individually. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.
- Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972).
- Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
- Following fixation in alcohol, each fetus was stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976). Fetuses were then examined for skeletal malformations and developmental variations. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter
general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
Statistics:
- Statistical analyses are described below.
Indices:
- Intrauterine data were summarized using two methods of calculation (group mean litter basis calculated as postimplantation loss and proportional litter basis calculated as summation per group (%).
- Postimplantation Loss/Litter = No Dead Fetuses, Resorptions (Early/Late)/Group / No Gravid Females/Group
- Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%) / No Litters/Group where Postimplantation Loss/Litter (%) = [No Dead fetuses, Resorptions (Early/Late)/Litter / No Implantation Sites/Litter] x 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; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis using the equation Summation per Group (%) = [(Sum of Viable Fetuses Affected / Litter (%)) / No Litters/Group] where Viable Fetuses Affected / Litter (%) = [(No Viable Fetuses Affected/Litter) / No Viable Fetuses/Litter] x 100.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related increased incidences of yellow material around the ventral trunk and urogenital areas were noted in the 1000 mg/kg/day group generally throughout the treatment period and were not considered to be adverse.
- Increased incidences of clear and red material around the mouth were noted in all test substance-treated groups at approximately 1 hour following dose administration when compared to the control group. These findings were generally noted throughout the treatment period. Because clear and red material around the mouth did not persist to the daily examinations on the following day they were not considered adverse.
- No other test substance-related clinical findings were noted approximately 1 hour following dose administration or at the daily examinations at any dosage level.
- Findings noted in the treated groups, including hair loss on the forelimbs and ventral trunk, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
- All females in the control, 250, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MATERNAL BODY WEIGHT CHANGES AND GRAVID UTERINE WEIGHTS
- Test substance-related, significantly (p<0.01) lower mean body weight gains were noted in the 1000 mg/kg/day group compared to the control group generally throughout the treatment period (gestation days 6-20). As a result, mean body weights in the 1000 mg/kg/day group were 5.4% to 11.8% lower than the control group during gestation days 17-20; the differences were significant (p<0.05 or p<0.01). Mean gravid uterine weight, net body weight, and net body weight gain in this group were also significantly (p<0.05 or p<0.01) lower than the control group.
- In the 500 mg/kg/day group, mean body weight gains were similar to the control group during gestation days 6-9. During gestation days 9-12 a significantly (p<0.01) higher mean body weight gain was noted in the 500 mg/kg/day group due to a significantly (p<0.01) higher mean body weight gain during gestation days 11-12 when compared to the control group. Test substance-related, significantly (p<0.05 or p<0.01) lower mean body weight gains were noted in this group compared to the control group during the remainder of the study (gestation days 12-15 and 15-20). However, the lower mean body weight gains noted in the 500 mg/kg/day group were not of sufficient magnitude to affect the overall treatment period (gestation days 6-20) and mean body weights in this group were similar to the control group throughout the study; therefore, these differences were not considered to be adverse. Mean net body weight, net body weight gain, and gravid uterine weight in the 500 mg/kg/day group were unaffected by test substance administration.
- Mean maternal body weights, body weight gains, net body weight, net body weight gain, and gravid uterine weight in the 250 mg/kg/day group 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, treatment-related
Description (incidence and severity):
- Test substance-related lower mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 1000 mg/kg/day group throughout the treatment period (gestation days 6-20). The differences from the control group were generally significant (p<0.05 or p<0.01) and corresponded to the lower mean body weight gains noted for these females.
- Mean maternal food consumption in the 250 and 500 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. Significantly (p<0.05) lower mean food consumption was noted in the 500 mg/kg/day group during gestation days 14-15 and 17-18. Because these differences were transient and did not affect the overall treatment interval, they 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:
no effects observed
Description (incidence and severity):
- At the scheduled necropsy on gestation day 20, no test substance-related internal findings were observed at dosage levels of 250, 500, and 1000 mg/kg/day.
- Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies
in the control group, 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

Other effects:
effects observed, treatment-related
Description (incidence and severity):
GESTATION DAY 20 LAPAROHYSTERECTOMY DATA
- Test substance-related effects on intrauterine survival were noted in the 1000 mg/kg/day group. The mean litter proportion of postimplantation loss (early and late resorptions) in the 1000 mg/kg/day group (18.5% per litter) was significantly (p<0.01) higher than the control group (3.1% per litter) and the value exceeded the maximum mean value in the WIL Research historical control data (11.3% per litter). As a result, a significantly (p<0.05 or p<0.01) lower mean number (12.7 per dam) and litter proportion (81.5% per litter) of viable fetuses was noted in this group when compared to the concurrent control group (15.1 per dam and 96.9% per litter, respectively) and the minimum mean value in the WIL Research historical control data (12.2 fetuses per dam and 88.7% per litter, respectively).
- Test substance-related effects on intrauterine growth were noted at dosage levels of 500 and 1000 mg/kg/day. In the 500 and 1000 mg/kg/day groups, test substance-related lower mean male (3.4 g and 2.6 g, respectively), female (3.2 g and 2.5 g, respectively), and combined-sex (3.3 g and 2.5 g, respectively) fetal body weights were noted compared to the concurrent control group values (4.0 g, 3.8 g, and 3.9 g, respectively); the differences were significant (p<0.01). The mean fetal body weights at 500 and 1000 mg/kg/day were also below the minimum mean male, female, and combined-sex fetal body weight values in the WIL Research historical control data (3.5 g, 3.4 g, and 3.4 g, respectively). Significantly (p<0.01) lower mean fetal body weights (male, female, and combined) were noted in the 250 mg/kg/day group compared to the control group. However, the magnitude (5.0% to 7.9% lower than the concurrent control group) of the differences was small and the values were within the WIL Research historical control ranges. Therefore, the differences in fetal body weights observed in the 250 mg/kg/day group were not considered to be test substance-related.
- Intrauterine survival at 250 and 500 mg/kg/day was unaffected by maternal test substance administration. Mean numbers of corpora lutea and implantation sites, mean fetal sex ratios, and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: lower mean bodyweight gains, bodyweights and food consumption at 1000 mg/kg bw/day

Results (fetuses)

External malformations:
effects observed, treatment-related
Description (incidence and severity):
EXTERNAL MALFORMATIONS AND VARIATIONS
- External malformations were noted in 0(0), 2(2), 1(1), and 52(12) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. Test substance-related external malformations were noted in the 1000 mg/kg/day group and consisted of increased mean litter proportions of fetal anasarca, cleft palate (confirmed skeletally for 1 of 2 fetuses as posterior portion of palatine plates not joined), and localized fetal edema (neck and/or thorax) compared to the concurrent control group.
- The differences from the concurrent control group were not statistically significant, but the mean litter proportions of these external malformations exceeded the maximum mean values in the WIL Research historical control data. Table 2 (attached) summarizes the absolute number of fetuses and mean litter proportions of the test substance-related external malformations noted in the 1000 mg/kg/day group.
- In the 1000 mg/kg/day group, fetus no. 24035-07 had a filamentous tail (skeletally all vertebrae posterior to sacral vertebra no. 4 were absent) and fetus no. 24048-13 had anal atresia and vertebral agenesis (anury; skeletally, all vertebrae posterior to sacral vertebra no. 2 were absent). These findings were noted in single fetuses and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the WIL Research historical control data ranges, therefore, they were not considered test substance-related.
- Fetus no. 24014-14 in the 500 mg/kg/day group had craniorachischisis (cranial vault and vertebral column were open through the sacral region) which consisted skeletally of dorsal aspects of cervical arch no. 1 through sacral arch no. 4 that were reflected more laterally than normal. In the 250 mg/kg/day group, fetus no. 24000-08 had a facial cleft (upper left rostral region) which skeletally consisted of absent nasal and premaxilla bones and misshapen maxilla bones and exencephaly with an open eyelid; skeletally this finding consisted of and absent frontal, parietal, interparietal, and supraoccipital bones. Fetus no. 24019-09 in the 250 mg/kg/day group had a cleft palate (entire length; not confirmed skeletally).
- The external malformations observed in the 250 and 500 mg/kg/day groups were noted for single fetuses, did not occur in a dose-related manner, and the mean litter proportions of these findings were not statistically significantly different from the concurrent control group and therefore they were not attributed to the test substance.
- There were no external developmental variations noted for fetuses in this study.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
SKELETAL MALFORMATIONS AND VARIATIONS
- Skeletal malformations were noted in 6(3), 3(3), 2(2), and 20(12) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively.
- Test substance-related skeletal malformations were noted in the 1000 mg/kg/day group and consisted of increased mean litter proportions of rib anomaly (small, fused, and/or reduced ossification of ribs), interrupted ossification of the rib(s), and bent limb bone(s) (radius, ulna, humerus, tibia, and/or fibula) compared to the concurrent control group. Although the mean litter proportions of these skeletal malformations were not statistically significantly different from the concurrent control group, the values exceeded the maximum mean values in the WIL Research historical control data. Table 4 (attached) summarizes the absolute number of fetuses and mean litter proportions of the test substance-related skeletal malformations noted at 1000 mg/kg/day. No other test substance-related skeletal malformations were noted at any dosage level.
- One fetus each in the 250 and 1000 mg/kg/day groups had a skull anomaly (basispheniod misshapen and supraoccipital bone absent, respectively). Two fetuses each in the 500 and 1000 mg/kg/day group had vertebral anomalies with or without associated rib anomalies. These skeletal malformations consisted of absent rib, arches, and centra; extra arches and ribs; malproportioned arches; malpositioned centra, arches, and ribs; fused ribs and arches; and forked ribs. In addition, 1 fetus in the 250 mg/kg/day group had sternoschisis (sternal band nos. 4 through 6 not joined) and 1 fetus in this same group had a costal cartilage anomaly (fused and malpositioned); costal cartilage anomalies were also noted for 6 fetuses in the control group. Because these skeletal malformations were noted for single fetuses, were observed similarly in the concurrent control group, and the mean litter proportions were not statistically significantly different from the concurrent control group and/or were within the WIL Research historical control data, they were not considered to be test substance-related.
- Test substance-related skeletal developmental variations were noted in the 500 and 1000 mg/kg/day groups. Increased mean litter proportions of 14th rudimentary rib(s), reduced ossification of the skull, reduced ossification of the vertebral arches, unossification of sternebra(e) nos. 5 and/or 6, unossification of sternebra(e) nos. 1, 2, 3, and/or 4, bent rib(s), and reduced ossification of the ribs and decreased mean litter proportions of ossification of cervical centrum no. 1 were noted in the 500 and 1000 mg/kg/day groups compared to the concurrent control group; the differences were generally significant (p<0.01) and the mean litter proportions were outside of the WIL Research historical control data ranges. Additionally, increased mean litter proportions of unossified hyoid, 14th full rib(s), 7th cervical rib(s), unossification of the entire sternum, pubis, ischium, and vertebral centra, and bent scapula(e) were noted at 1000 mg/kg/day when compared to the concurrent control group; all values were significant (p<0.01) with the exception of 14th full rib(s), 7th cervical rib(s), and vertebral centra unossified, and the mean litter proportions of these findings exceeded the maximum mean values in the WIL Research historical control data ranges. The majority of the test substance-related skeletal developmental variations noted in the 500 and 1000 mg/kg/day groups consisting of reduced ossification or unossified bones were indicative of developmental delay and considered secondary to the reduced fetal weights noted at these dosage levels. Table 5 (attached) summarizes the absolute number of fetuses and mean litter proportions of the test substance-related skeletal developmental variations noted in the 500 and 1000 mg/kg/day groups.
- Other skeletal developmental variations observed in the test substance-treated groups consisted of 27 presacral vertebrae, sternebra(e) malaligned (slight or moderate), and reduced ossification of the 13th rib(s). These findings occurred infrequently or at a frequency similar to the concurrent control group, did not occur in a dose-related manner, and/or the mean litter proportions were within the WIL Research historical control data range, and therefore were not considered to be test substance-related.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
VISCERAL MALFORMATIONS AND VARIATIONS
- Visceral malformations were noted for 0(0), 2(2), 0(0), and 12(8) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. A test substance-related visceral malformation noted in the 1000 mg/kg/day group consisted of a right-sided aortic arch (aortic arch and descending aorta course to the right of the vertebral column; right carotid and right subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk] left carotid and left subclavian arteries arose from a common vessel from the aortic arch). Although the mean litter proportion of this finding was not statistically significantly different from the concurrent control group; the value exceeded the maximum mean value in the WIL Research historical control data. No other test substance-related visceral malformations were noted for fetuses in this study.
- In the 1000 mg/kg/day group, coarctation of the pulmonary trunk was noted for fetus nos. 23982-11 and 24012-08 (also noted with transposition of the great vessels), cartilaginous bands were absent from the entire length of the trachea for fetus no. 23972-17, and vestigial aortic arch was noted for fetus no. 23994-03. Interventricular septal defect was noted for 1 and 2 fetuses in the 250 and 1000 mg/kg/day groups, respectively, and coarctation of the aortic arch was noted for 1 fetus each in these same groups. Also in the 250 mg/kg/day group, fetus no. 24034-12 had lobular dysgenesis of the lungs (1 lobe present) and situs inversus (the liver, stomach, pancreas, spleen, kidneys, adrenals, and intestine were laterally transposed). These visceral malformations were noted for 1-2 fetuses per group, the mean litter proportions were not statistically significantly different from the concurrent control group, and/or the mean litter proportions of these findings were within the WIL Research historical control data ranges. Therefore, these visceral malformations were not considered to be test substance-related.
- Test substance-related visceral developmental variations (see Table 3, attached) noted in the 1000 mg/kg/day group include higher mean litter proportions of small and pale spleen and major blood vessel variation (right carotid and right subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk]) compared to the concurrent control group. Although the mean litter proportions of these findings were not statistically significantly different from the concurrent control group; they exceeded the maximum mean values in the WIL Research historical control data and were therefore attributed to maternal test substance administration.
- All other visceral developmental variations noted in the test substance-treated groups were noted at similar frequencies in the concurrent control group, did not occur in a dose-related manner, and/or the mean litter proportions of the findings were not statistically significantly different from the concurrent control group and were within the WIL Research historical control data ranges and therefore were not considered test substance-related.
- Renal papilla(e) not fully developed (Woo and Hoar Grade 1) were noted for 1, 3, and 2 fetuses in the 250, 500, and 1000 mg/kg/day groups, respectively. In addition, 1 fetus (no. 23967-12) in the 500 mg/kg/day group had a swollen liver (all lobes). These findings were not classified as either malformations or developmental variations, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently and/or in a manner that was not dose-related.
Details on embryotoxic / teratogenic effects:
SUMMARY OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
- The numbers of fetuses (litters) available for morphological evaluation were 377(25), 348(25), 373(25), and 318(24) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. Malformations were observed in 6(3), 6(6), 2(2), and 72(20) fetuses (litters) in these same respective dose groups.
- A significantly (p<0.01) higher total mean litter proportion of fetal malformations was observed in the 1000 mg/kg/day group (26.1% per litter) compared to the control group (1.7% per litter). This was due to the higher percent per litter of external (19.2% per litter), soft tissue (3.4% per litter), and skeletal (8.1% per litter) malformations noted at 1000 mg/kg/day compared to the concurrent control group values (0.0%, 0.0%, and 1.7% per litter, respectively); only the difference for external malformations was significant (p<0.05). Higher incidences of external (fetal anasarca, cleft palate, and localized fetal edema), visceral (right-sided aortic arch), and skeletal (rib anomaly, interrupted ossification of the rib[s], and bent limb bone[s]) fetal malformations were noted in the 1000 mg/kg/day group.
- Significantly (p<0.01) higher total mean litter proportions of fetal developmental variations were observed in the 500 and 1000 mg/kg/day groups (78.4% and 100.0%, respectively) compared to the concurrent control group (45.3% per litter). This was due to the significantly (p<0.01) higher percent per litter of skeletal variations in the 500 and 1000 mg/kg/day groups (77.7% and 100.0% per litter, respectively). Test substance-related increased incidences of skeletal developmental variations (ossification of 14th rudimentary rib[s], reduced ossification of the skull, reduced ossification of the vertebral arches, unossification of sternebra[e] nos. 5 and/or 6, unossification of sternebra[e] nos. 1, 2, 3, and/or 4, bent rib[s], and reduced ossification of the rib[s]) and decreased incidences of ossification of cervical centrum no. 1 were noted at 500 and 1000 mg/kg/day. Additionally, increased incidences of skeletal developmental variations consisting of unossified hyoid, 14th full rib(s), unossification of the entire sternum, 7th cervical rib(s), unossified pubis, unossified ischium, unossified vertebral centra, and bent scapula(e) were noted at 1000 mg/kg/day. The skeletal developmental variations noted in the 500 and 1000 mg/kg/day groups consisting of reduced ossification or unossified bones were indicative of developmental delay and considered secondary to the reduced fetal weights noted at these dosage levels. Additionally, test substance-related visceral developmental variations consisting of increase in the mean litter proportions of small and pale spleen and major blood vessel variation were noted at 1000 mg/kg/day.
- No test substance-related fetal malformations were noted in the 250 or 500 mg/kg/day groups and no test substance-related developmental variations were noted in the 250 mg/kg/day group.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks:
maternal dose
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: see discussion below

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

ANALYSES OF DOSING FORMULATIONS

- The mean concentrations of the initial 50, 100, and 200 mg/mL formulations prepared on 08-Aug-2014 and the mean concentration of the final 50 mg/mL formulation prepared on 21-Aug-2014 were between 71.5% and 83.9% of the target concentration; all other samples were between 90.5% and 92.8% of the target concentration.

- The results of these analyses were consistent with the results obtained during the method validation 70-day storage assessment; i.e., formulations at lower prepared concentrations have lower mean percent recovery following prolonged storage (Haas, 2015, WIL-168211).

- For logistical reasons (i.e., sample shipment and method development times) sample analyses in this study were conducted approximately 26 to 45 days after formulation and dosing. However, because all formulations were used for dosing within 8 days of preparation, it is believed that the animals on this study received the intended dose concentration/dosage levels.

- The test substance was not detected in the vehicle formulation that was administered to the control group (Group 1).

- Analyses of dosing formulations are summarised in Table 1 (attached).

Applicant's summary and conclusion

Conclusions:
Maternal toxicity was evidenced at 1000 mg/kg/day by lower mean body weight gains, body weights, and food consumption. No evidence of maternal toxicity was noted at 250 or 500 mg/kg/day. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Developmental toxicity was noted at 500 and 1000 mg/kg/day as evidenced by lower mean fetal body weights, which, in conjunction with a lower number of viable fetuses, correlated with a lower mean gravid uterine weight in the 1000 mg/kg/day group. Higher mean litter proportion of postimplantation loss with a corresponding lower mean number and litter proportion of viable fetuses were also noted at 1000 mg/kg/day. In addition, test substance-related fetal malformations were noted at 1000 mg/kg/day and test substance-related developmental variations were noted at 500 and 1000 mg/kg/day. Intrauterine growth in the 250 mg/kg/day group and survival in the 250 and 500 mg/kg/day groups were unaffected by test substance administration. Based on these results, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.
Executive summary:

GUIDELINE

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, and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity. The protocol was designed in general accordance with EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August, 1998 and the OECD Guidelines for the Testing of Chemicals, Guideline 414, Prenatal Developmental Toxicity Study, January 2001.

 

METHODS

The test substance in the vehicle (peanut oil) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 250, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

RESULTS

All females in the control, 250, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. Test substance-related, nonadverse increased incidences of clear and red material around the mouth were noted in the 250, 500, and 1000 mg/kg/day group approximately 1 hour following dose administration and increased incidences of yellow material around the ventral trunk and urogenital areas were noted in the 1000 mg/kg/day group generally throughout the treatment period. There were no other test substance-related clinical findings noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

 

Test substance-related lower mean body weight gains with corresponding lower mean food consumption were noted in the 1000 mg/kg/day group throughout the treatment period. In addition, lower mean body weights, net body weight, and net body weight gain were observed at this dosage level.

 

In the 500 mg/kg/day, mean body weight gains were similar to the control group during gestation days 6-9 and then lower than the control group during the remainder of the treatment period (gestation days 9-12, 12-15, and 15-20). However, the lower mean body weight gains noted in this group were not of sufficient magnitude to affect the overall treatment period (gestation days 6-20) or mean body weights and therefore were not considered to be adverse. There were no test substance-related effects on mean body weight gains at 250 mg/kg/day or mean body weights, net body weights, net body weight gains, food consumption, or gravid uterine weights at 250 and 500 mg/kg/day.

 

There were no test substance-related macroscopic findings noted at any dosage level.

 

Mean fetal body weights in the 500 and 1000 mg/kg/day groups were up to 15.8% and 35.9% lower, respectively, than the control group. The lower mean fetal weights corresponded to the lower mean gravid uterine weight in the 1000 mg/kg/day group; mean gravid uterine weight in the 500 mg/kg/day group was similar to the control group. In the 1000 mg/kg/day group, a higher mean litter proportion of postimplantation loss and a corresponding lower mean number and mean litter proportion of viable fetuses were noted compared to the control group. Intrauterine growth at 250 mg/kg/day and survival at 250 and 500 mg/kg/day were unaffected by maternal test substance administration.

 

In the 1000 mg/kg/day groups, test substance-related higher incidences of external (fetal anasarca, cleft palate, and localized fetal edema around the neck and/or thorax), visceral (right-sided aortic arch), and skeletal (rib anomaly, interrupted ossification of the rib[s], and bent limb bone[s]) fetal malformations and visceral developmental variations (small and pale spleen and major blood vessel variation) were noted compared to the control group. In addition, test substance-related skeletal developmental variations were noted at 500 and 1000 mg/kg/day. Higher incidences of 14th rudimentary rib(s), reduced ossification of the skull and vertebral arches, unossification of sternebra(e) nos. 5 and/or 6 and sternebra(e) nos. 1, 2, 3, and/or 4, bent rib(s), and reduced ossification of the rib(s), in addition to lower incidences of ossification of cervical centrum no. 1, were noted in the 500 and 1000 mg/kg/day groups when compared to the control group. In addition, higher incidences of 14th full rib(s), unossification of the hyoid, entire sternum, pubis, ischium, and vertebral centra, 7th cervical rib(s), and bent scapula(e) were noted at 1000 mg/kg/day. The aforementioned skeletal developmental variations were considered test substance-related and secondary to the reduced fetal weights noted at these dosage levels. No test substance-related fetal malformations were noted at 250 and 500 mg/kg/day and no test substance-related developmental variations were noted at 250 mg/kg/day.

 

CONCLUSIONS

Maternal toxicity was evidenced at 1000 mg/kg/day by lower mean body weight gains, body weights, and food consumption. No evidence of maternal toxicity was noted at 250 or 500 mg/kg/day. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Developmental toxicity was noted at 500 and 1000 mg/kg/day as evidenced by lower mean fetal body weights, which, in conjunction with a lower number of viable fetuses, correlated with a lower mean gravid uterine weight in the 1000 mg/kg/day group. Higher mean litter proportion of postimplantation loss with a corresponding lower mean number and litter proportion of viable fetuses were also noted at 1000 mg/kg/day. In addition, test substance-related fetal malformations were noted at 1000 mg/kg/day and test substance-related developmental variations were noted at 500 and 1000 mg/kg/day. Intrauterine growth in the 250 mg/kg/day group and survival in the 250 and 500 mg/kg/day groups were unaffected by test substance administration. Based on these results, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.