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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20-5-2016 to 25-7-2016 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
- Lot No. F534801GB
- Exp. date: 11 Dec 2016
- Colorless, clear liquid

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
Hra:(NZW)SPF
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA
- Age at study initiation: The animals were approximately 7 months old upon receipt.
- Weight at study initiation: 2922 - 3949 g
- Housing: All rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). Nesting material was not required because the females were euthanized prior to the date of expected parturition. 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. Kale (1 leaf at each occasion) was provided to each animal daily for environmental enrichment and to aid in maintaining the animal's gastrointestinal health, beginning upon animal receipt and continuing throughout the duration of the study.
- Diet: The basal diet used in this study, PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322, was a certified feed with appropriate analyses performed by the manufacturer. The basal diet was offered in 25-g increments 3 times per day on the day of arrival and in increased amounts over the next few days, until the animals gradually achieved ad libitum status prior to the dose administration period; basal diet was offered ad libitum thereafter.
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum during the study.
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% carboxymethylcellulose (medium viscosity), 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily as single formulations for each dosage level and maintained on wet ice, protected from light. The test substance formulations were stirred continuously on wet ice throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 10, 30 and 80 mg/mL (corresponding to dosage levels of 0, 50, 150 and 400 mg/kg/day)
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the 10, 30, and 80 mg/mL dosing formulations and from the middle stratum of the control group dosing formulations prepared on on the first, approximate middle, and last days of preparation on which all groups were dosed. Analysis was performed using a validated gas chromatography method using flame ionization detection. The analyzed concentrations was 85% to 115% of the target concentration.
Details on mating procedure:
The time-mated rabbits were received on gestation day 2, 3, or 4; a breeding record was provided by the supplier.
Duration of treatment / exposure:
gestation days 7 through 28
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were selected based on the range-finding stud (see any other information on materials and methods)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS
All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from the day of receipt through gestation day 29 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration.

BODY WEIGHT
Individual maternal body weights were recorded on gestation days 0 (by supplier under conditions that were not compliant with GLPs, but in accordance with the supplier’s SOPs), 5, and 7-29 (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 7-10, 10-13, 13-21, 21-29, and 7-29.

FOOD CONSUMPTION
Individual food consumption was recorded on gestation days 5-29. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

POST-MORTEM EXAMINATIONS
The laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rabbits were euthanized on estation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. 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 co related with the antemortem observations, and any abnormalities were recorded. 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Gravid uterine weight was collected and net body weight (the gestation day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
- Number of corpora lutea: The number of corpora lutea on each ovary was recorded.
- Number of implantations: 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. 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. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in
10% ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
- Fetal examinations were performed blind to treatment group.
- External examinations: Each viable fetus was examined externally, individually weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. 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. Crown-rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions, and the tissues were discarded.
- Soft tissue examinations: 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. The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development
- Skeletal examinations: Following fixation in alcohol, each fetus was stained with Alizarin Red S8 and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
- Head examinations: 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. 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.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group.
- Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group.
- Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.
Indices:
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

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
Historical control data:
yes

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 findings 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 decreased defecation and brown material and/or 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.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy on gestation day 29.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 400 mg/kg/day group, an absence of a mean body weight gain (0 g) was noted on the first day of dose administration (gestation day 7-8) and resulted in a 78.9% lower mean body weight gain in this group compared to the control group during gestation days 7-10 (57 g, 54 g, and 64 g in the control, 50, and 150 mg/kg/day groups, respectively, compared to 12 g in the 400 mg/kg/day group). Mean body weight gains in the 400 mg/kg/day group were similar to the control group for the remainder of the treatment period (gestation days 10-29). As a result of the lower mean body weight gain at the beginning of the treatment period, a lower mean body weight gain was noted at 400 mg/kg/day compared to the control group when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse.
Mean maternal body weight gains in the 50 and 150 mg/kg/day groups and mean body weights, net body weights, net body weight gains, and gravid uterine weights in the 50, 150, and 400 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, treatment-related
Description (incidence and severity):
In the 400 mg/kg/day group, lower mean food consumption was noted during gestation days 7 -10 compared to the control group; mean food consumption in this group was similar to the control group for the remainder of the treatment period (gestation days 10-29). As a result of the lower mean food consumption at the beginning of the treatment period, lower mean food consumption was noted in the 400 mg/kg/day group compared to the control group when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences at 400 mg/kg/day were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse.
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50 and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
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
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on gestation day 29, no test substance-related internal findings were observed at dosage levels of 50, 150, and 400 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed
Other effects:
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.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal systemic toxicity & maternal developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related external malformations were noted for fetuses in this study. In the 150 mg/kg/day group, one fetus was noted with omphalocele (a portion of the liver protruded through an opening in the umbilicus, remnants of a membranous sac). The aforementioned malformation noted at 150 mg/kg/day occurred in a single fetus, did not occur in a dose-related manner, and the mean litter proportion was not statistically significantly different from the concurrent control group and was within the test lab historical control data range (version 2016.01); therefore, it was not considered test substance-related. No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related skeletal malformations were noted for fetuses at any dosage level. Vertebral anomaly with or without an associated rib anomaly (extra or fused ribs; extra and/or malpositioned arches; extra, malpositioned, absent, small, fused, and/or misshapen centra) was noted for 2 and 1 fetuses in the 50 and 150 mg/kg/day groups, respectively. The aforementioned malformation at 50 and 150 mg/kg/day occurred infrequently, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the Charles River Ashland historical control data range; therefore, it was not considered test substance-related.
- 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:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related visceral malformations were observed for fetuses at any dosage level. Lobular agenesis of the lungs (right accessory lobe absent) was noted for and 3 fetuses in the 50 and 150 mg/kg/day groups, respectively. Because this finding occurred infrequently, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the Charles River Ashland historical control data range, this finding was not considered test substance-related. In the control group, one fetus was noted with an absent left kidney and ureter and one fetus was noted with an absent right thyroid gland.
- No test substance-related visceral 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.
- A distended stomach was noted for one fetus in the 50 mg/kg/day group. This finding was not classified as either a malformation or developmental variation, was not considered to be test substance-related because it occurred infrequently and in a manner that was not dose-related.
Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 219(25), 214(24), 199(25), and 214(24) in the control, 50, 150, and 400 mg/kg/day groups, respectively. Malformations were observed in 2(1), 4(4), 5(4), and 0(0) fetuses (litters) in these same respective dosage groups and were considered spontaneous in origin.

Effect levels (fetuses)

Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion