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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 October 2017 - 1 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August, 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May, 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF: The Japanese Ministry of Agriculture, Forestry and Fisheries Notification of 12 NohSan-8147, Guideline 2-1-18, Teratogenicity study
Version / remarks:
November, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Species and Sex: Rabbits, time-mated females
Strain: NZW rabbits
Supplier: Covance Research Products, Inc. (CRP), Greenfield, Indiana, USA
Age and Weight at Study Start: Sexually mature adults weighing 2800-3200 g.

Health Status and Acclimation:
Upon arrival all animals were acclimated to the laboratory for approximately 5 days prior to the start of test material administration. Upon arrival and once during the acclimation period, each animal was evaluated by trained veterinarian to determine the general health status and acceptability for study purposes.

Housing:
Upon arrival and after assignment, animals were housed one per cage in stainless steel and plastic cages. Cages had perforated plastic floors and were suspended above catch pans with absorbent non-contact bedding. Cages contained a J-type stainless steel feeder and a pressure activated lixit valve-type stainless steel watering system.

The following environmental conditions were targeted in the animal room from the day of arrival until necropsy:

Temperature: 20°C with a range of 16°C-22°C (targeted) 19.9°C with a range of 19°C-21°C (actual)
Humidity: 50% with a range of 40-70% (targeted) 50.3% with a range of 49.2-57.7% (actual)
Air Changes: 10-15 times/hour ( targeted average) 11.6 times/hour (actual average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Enrichment:
Enrichment for animals was given from the day of arrival until necropsy. The enrichment included an elevated resting platform, a variety of stainless steel objects attached inside the cage, and a cardboard tray for manipulation.

Feed and Water:
A stepwise increase in amount of daily feed allotted was implemented to aid in avoiding gastrointestinal disturbances during the acclimation period. Upon receipt, rabbits received approximately 62.5 g of LabDiet 5325 (PMI Nutrition International, Richmond, Indiana) in pelleted form. The amount of feed was increased up to the full daily amount of approximately 125 g the following day. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test material was administered daily by oral gavage from GD 7-27.
Analytical verification of doses or concentrations:
no
Remarks:
No analyses were performed because the test material was administered neat (undiluted). Stability was not applicable to this study due to utilizing neat test material.
Details on mating procedure:
Sexually mature virgin females were naturally mated with one buck of the same strain at the supplier. The observed day of breeding was considered GD 0. GD 0 body weights and records of mating pairs were provided by the supplier and maintained in the study record. Rabbits arrived in the laboratory on GD 1 or 2.
Duration of treatment / exposure:
Rabbits were administered D6 on GD 7-27
Frequency of treatment:
Test material was administered daily by oral gavage from GD 7-27
Duration of test:
Test material administration began on November 13, 2017 and the last group of rabbits was necropsied on December 13, 2017.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Dose Volume: 1.0204 ml/kg
Dose / conc.:
100 mg/kg bw/day
Remarks:
Dose Volume: 0.1020 ml/kg
Dose / conc.:
300 mg/kg bw/day
Remarks:
Dose Volume: 0.3061 ml/kg
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Dose Volume: 1.0204 ml/kg
No. of animals per sex per dose:
24 number of rabbits/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 24 time-mated female NZW rabbits were administered D6 via gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day on GD 7-27. The rabbits were ordered and mated in six different replicates from the supplier to stagger caesarean sections over a period of two weeks.

Dose Levels and Justification
Dose levels for this study (0, 100, 300, or 1000 mg/kg/day on GD 7-27) were selected on the basis of the developmental toxicity probe study discussed previously (Johnson et al., 2018). The high-dose of 1000 mg/kg/day represented a limit dose as defined in the test guideline. The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rabbits.

Dose Preparation
The test material was administered neat (undiluted). Dose volumes were calculated using the most current body weight. Control animals were gavaged with 1.0204 ml of water/kg body weight, which was the volume equal to the largest gavage volume given treated animals.

Examinations

Maternal examinations:
Daily Observations:
A cage-side examination was conducted twice daily, approximately at the same time each day. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. For animals showing indications of premature delivery, the delivered fetuses were counted and examined to the extent possible.

Clinical Observations
Clinical observations were conducted on all animals at least once daily. Animals were observed approximately one hour after dosing. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Body Weights/Body Weight Gains
Body weights were recorded on GD 0 by the supplier, GD 4, daily during test material administration, and on GD 28.

Feed Consumption
Daily feed consumption was recorded and statistically analyzed for all animals from GD 4-28.
Ovaries and uterine content:
On GD 28, all surviving females (not fasted) were euthanized via intravenous injection of Beuthanasia-D (Henry Schein Animal Health, Dublin Ohio), and a limited gross pathological examination (necropsy) was performed. The sequence of the maternal necropsies was counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification.
A detailed examination of the reproductive tract was performed and the number and position of implantations, viable fetuses, dead fetuses and resorptions were recorded. Resorptions were classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a “dead fetus” indicated a very recent death as evidenced by a lack of external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea was counted. The uteri of females lacking visible implantations were stained with a 10% aqueous solution of sodium sulfide (based on Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.
Fetal examinations:
The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. The stomach, liver (with gallbladder) and kidneys were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded, and the weight of the liver (with incised gallbladder), kidneys and gravid uterus were recorded. The ratios of liver and kidney weights to terminal body weight were calculated. Representative sections of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of tissues was not conducted. Transponders were removed and placed in bags with the tissues.

All fetuses were weighed, and given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were then given a secondary dose of euthanasia solution via sublingual oral administration of Fatal Plus (Schering Corporation, Kenilworth, New Jersey). All fetuses were given a visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984). The visceral examination included observations of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidneys (sectioned), adrenal glands, ureters, bladder, and reproductive organs. The fetuses were sexed by examination of the gonads. Approximately one half of the fetuses in each litter were randomly selected for craniofacial examination. The heads of these fetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965). All fetuses were preserved in alcohol, eviscerated and stained with Alizarin Red S in order to visualize ossified bone (Dawson, 1926). After staining, skeletons were cleared and a thorough evaluation of the fetal skeleton was conducted.
Statistics:
The litter, rather than the pup, was considered as the experimental unit. Maternal body weights, maternal body weight gains, organ weights (absolute and relative with the exception of only absolute weight for gravid uterus), fetal body weights and feed consumption were evaluated by Bartlett’s test (alpha = 0.01; Winer, 1971) for homogeneity of variance. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with Bonferroni's correction (Miller, 1966) was performed, respectively.
Feed consumption values were excluded from analysis if the feed was spilled or scratched.
Statistical analyses were conducted on average percent litter response for pre- and post-implantation loss and fetal alterations. Frequency of pre- and post-implantation loss (calculations shown below), and fetal alterations (if any) were analyzed using a censored Wilcoxon test (Haseman and Hoel, 1974) with Bonferroni’s correction applied when the incidence was greater than 5%.
The number of corpora lutea, implantations, and litter size were evaluated using a nonparametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction.
Pregnancy rates were analyzed using the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni’s correction.
Fetal sex ratios were analyzed using a binomial distribution test.
Non-pregnant females were excluded from the appropriate analyses.
Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969).
Both Dunnett’s test and Bonferroni’s correction corrected for multiple comparisons to the control and were reported at the corrected alpha level.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Examinations performed on all animals revealed no treatment-related findings.
One animal in the 300 mg/kg/day group delivered early and had pups present in her cage on the morning of GD 28 which was considered unrelated to treatment due to the isolated occurrence. Normal necropsy and fetal exams were performed for this animal to the extent possible except a gravid uterine weight was not recorded. Another animal in the 300 mg/kg/day group was removed from the study and euthanized on GD 26 due to a mechanical injury sustained from a gavage incident.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related differences in the amount of feed consumed by any treated groups when compared to their respective controls. Statistically-identified differences were considered spurious and unrelated to treatment due the minimal difference from control (≤4.2%).
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
Neuropathological findings:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Percent post-implantation loss in the 1000 mg/kg/day groups was slightly higher than controls and reached statistical significance. This increase in post-implantation loss was deemed spurious and unrelated to treatment as:
1) postimplantation loss in the 1000 mg/kg/day group was similar to recent historical controls,
2) postimplantation loss in the control group was lower than recent historical control
3) the postimplantation loss was represented by litters with single resorptions.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
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
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related skeletal alterations in any dose group. The incidence of one skeletal variation (DO sternebrae) was higher in the 300 and 1000 mg/kg/day dose groups compared to controls. This finding was deemed to be unrelated to treatment as it was within (litter) or similar (fetuses) the range of recent historical control values. Furthermore, this finding is considered to be a non-adverse variation as it is among the most common skeletal variations encountered in this type of guideline developmental toxicity study (Carney and Kimmel, 2007). In both rodents and rabbits, the sternebrae is an area that ossifies rapidly during late gestation (Carney and Kimmel, 2007; Fritz, 1975). Therefore, variable ossification of these structures is normal when necropsy occurs on GD 28. In addition, high background incidence is common due to the laboratory scoring criteria which dictates that the call of delayed ossification be made when >50% of sternebrae numbers 1,2,3,4 and 6 are unossified or sternebra 5 is completely unossified. Delays in ossification are not expected to persist in the postnatal animal due to high remodeling capacity of the skeleton (Carney and Kimmel, 2007; Holmbeck and Szabova, 2006).
Incidental findings bearing no relationship to treatment included the malformations misaligned thoracic centra, forked ribs, and thoracic hemivertebra and the variations delayed ossification (DO) hyoid, crooked hyoid, DO dentoid, DO sternebrae, fused sternebrae, irregular pattern of ossification sternebrae, DO thoracic centra, and DO pubis. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically-identified visceral alterations in any dose group. Incidental findings bearing no relationship to treatment included the malformations ventricular wall defect and missing gall bladder, and the variations missing caudal lung lobe, hemorrhage thymus, supernumerary hepatic liver lobule, hypoplastic spleen, particulate material kidney, bifurcated renal vein, retrocaval ureter, and paraovarian cyst. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
Details on embryotoxic / teratogenic effects:
No indication of embryo/fetal toxicity or teratogenicity.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects. The incidence of one skeletal variation (DO sternebrae) at 300 and 1000 mg/kg/day dose groups was deemed to be unrelated to treatment.
Remarks on result:
other: No treatment related effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The administration of D6 by gavage up to and including the limit dose of 1000 mg/kg/day, resulted in no treatment-related maternal toxicity and no indication of embryo/fetal toxicity or teratogenicity. Therefore, under the conditions of this study, NOAEL for maternal toxicity and developmental toxicity was the limit dose of 1000 mg/kg/day.
Executive summary:

The maternal and developmental toxicity of D6 in NZW rabbits following repeated oral gavage administration was evaluated. Groups of 24 time-mated female rabbits were administered 0, 100, 300, or 1000 mg/kg/day on gestation day (GD) 7-27. No analyses of concentration verification, homogeneity, or stability were conducted because the test material was administered neat (undiluted). In-life parameters evaluated for all groups included: clinical observations, body weight, body weight gain, and feed consumption. On GD 28 all surviving rabbits were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed, and examined for external and visceral alterations. The heads were examined for craniofacial alterations by serial sectioning in approximately one half of the fetuses in each litter, and skeletal examinations were performed on all fetuses.

Gavage administration of D6, up to and including the limit dose of 1000 mg/kg/day, resulted in no treatment-related maternal toxicity and no indication of embryo/fetal toxicity or teratogenicity. Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity was the limit dose of 1000 mg/kg/day.