Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-27 to 2016-03-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada Inc., St-Constant, Québec, Canada
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 196 - 263 g
- Housing: Animals were housed individually in stainless steel cages equipped with an automatic watering system. Following group assignment, all cages were clearly labeled with a color-coded cage card indicating at least the following: study number and animal number (including gender and group number).
- Diet: A standard certified commercial chow (Envigo (formerly Harlan) Teklad Certified Global Rodent Diet #2018C) was provided to the animals ad libitum.
- Water: Municipal tap water (which was exposed to ultraviolet light and purified by reverse osmosis) was provided to the animals ad libitum, via an automatic watering system or water bottles.
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 3 °C
- Humidity: 50 +/- 20 %
- Air changes: 10 - 15 per hour
- Photoperiod: 12 / 12 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item and reference item/vehicle dosing formulations were prepared at least weekly. The appropriate amount of test item was weighed and transferred into a container (or weighed directly into the container). The vehicle (corn oil) based on its ability to solubilize the test item) was added and QS’d followed by stirring. When necessary, an appropriate homogenizer was used to assure a homogenous suspension was prepared. The dosing formulations were stored at room temperature prior to administration. The results for specific gravity measured on the first formulations preparation were 0.9201, 0.9195, 0.9189 and 0.9160 for Group 1, 2, 3 and 4, respectively.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was chosen based on its ability to solubilize the test item.
- Concentration in vehicle: 3.75; 12.5; 62.5 mg/mL
- Amount of vehicle: 4 mL/kg bw/day
- Lot/batch no.: MKBS6944V/MKBV2080V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To verify the concentration and homogeneity of the test item in the formulations, representative samples (5 mL, in duplicate) were taken from the top, middle, and bottom of each concentration at the start of the study, and from the middle of a preparation conducted towards the end of the treatment period. An additional 15-mL sample was collected from the middle of each concentration at the start of the study for density measurement. The samples for concentration and homogeneity were stored frozen in a freezer (set to maintain -20°C). One of the two sets of the duplicate samples was shipped on dry ice to CiToxLAB Hungary. The second set of the duplicates were retained frozen at CiToxLAB North America. The analysis was performed by the analytical laboratory by gas chromatography (15/347-316AN) within the expected 1-month stability data. An acceptance criterion for the formulation was ± 15 % of nominal.
Details on mating procedure:
- Animals had been mated at Charles River Laboratories on the day prior to shipping, and on the following day, those females confirmed as mated (with vaginal plugs) were shipped to CiToxLAB North America. The day of receipt (day following mating) was considered Day 0 of the study.
- Proof of pregnancy: vaginal plug; referred to as day 0 of pregnancy
Duration of treatment / exposure:
16 days; from gestation day (GD) 5 to 20 (incl.)
Frequency of treatment:
daily
Duration of test:
Treatment from gestation day 5 to 20 (incl.); euthanasia at GD 21
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on available data and previous experiments performed in rats, including Harlan Study Number D53274 (Combined Repeated Dose Toxicity 28-day / Reproduction/Developmental Toxicity Screening Test, OECD 422). In that study, Han Wistar rats were administered test item at dose levels of 40, 125 and 400 mg/kg bw/day. All males survived until the scheduled necropsy. The No-Observed-Adverse-Effect-Level (NOAEL) was determined to be 400 mg/kg bw/day (males), and 40 mg/kg bw/day in females; the reproduction and development No Observed Effect Level (NOEL) was also 40 mg/kg bw/day. Mortality of females was observed at the end of the gestation period at dose levels of 125 and 400 mg/kg bw/day (2 and 8 females, respectively), possibly due to complications during the last days of pregnancy and/or difficult parturition. No clinical effects were observed at 40 and 125 mg/kg bw/day. Moderately decreased food intake was noted at 400 mg/kg bw/day (both males and females) at the beginning of the prepairing period, but was reversible thereafter. A body weight loss was noted at 400 mg/kg bw/day (both genders) at dosing start, but it recovered. There were no adverse test item-related histopathological changes. Therefore, a high dose level of 250 mg/kg bw/day was selected for this study. The low and mid dose were selected to provide a graded dose response.

- Rationale for animal assignment: Following arrival, animals were randomly assigned to dose groups by stratification based on body weights. For logistical reasons, the animals were assigned to replicate subgroups, which initiated dosing on consecutive days.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality checks were recorded concomitantly with the cage-side observations during all phases of the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily during the study, except on the day of arrival and necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 5, 6, 9, 12, 15, 18 and 21 (prior to Caesarean section).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined as follows: Gestation Days 0-3, 3-5, 5-6, 6-9, 9-12, 12-15, 15-18, and 18-21.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Numerical data obtained during the conduct of the study were subjected to calculation of group means and standard deviations and reported. The following statistical group comparison did not include groups with less than three observations or with zero variance. For each data set with more than two groups to compare, a one-way analysis of variance (ANOVA) was performed and the residuals tested for normality using a Shapiro-Wilk test. If the distribution of the residuals were significantly different from a normal one (p ≤ 0.05), the results from the ANOVA were discarded and a second ANOVA performed using square-root transformed data. When a data set included negative values, the absolute lowest observed value was added to the data before the transformation. The normality distribution of the residuals from the second ANOVA was tested using a Shapiro-Wilk test. When the Shapiro-Wilk test was not significant (p > 0.05) a Levene test was performed on the residuals to assess the homogeneity of the group variances. If differences between group variances were not found to be significant (p > 0.05) then the results from the related ANOVA were retained. When significant differences among the group means were indicated by the ANOVA overall F test (p ≤ 0.05), a Dunnett test was used to perform the group mean comparisons between the control treated group and each test item treated group. When the Shapiro-Wilk test on the residuals of the second ANOVA was significant or when heterogeneous group variances (p ≤ 0.05) were revealed by the Levene test, then the ANOVA results were discarded and the groups compared using a non-parametric Kruskal Wallis test. When the Kruskal Wallis test was significant (p ≤ 0.05), a Dunn test was used to perform the pairwise group comparisons between the control treated group and each test item treated group.
Indices:
Group incidence results were presented for each considered parameter. All groups with less than four observations were excluded from the following statistical comparison. For more than two groups to compare, an overall test (including all groups) was performed using the Fisher exact test . If the overall test was significant (p ≤ 0.05), the pairwise group comparison of the control group with each of the other test item treated groups were done using Fisher’s exact test.
Indices:
- external, internal and skeletal findings
- macroscopic findings

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In animals surviving to study termination, there were no observed adverse clinical signs considered related to the administration of the test item at any dose level; however, clinical signs including severely reduced activity, cold to touch, lying on cage floor, and decreased respiration rate, occurred on one high dose animal (4520). Other clinical observations that included, but were not limited to skin red, skin dry, fur stained red, skin scab were noted in all groups, prior to, during or post-dosing and were not considered to be test item-related. Animal 4524 (High Dose) was observed weak with piloerection, hunched back, cold to touch, skin turgor slow and ptosis following dosing on GD 8 and/or prior to dosing on GD 9. These signs, were not considered test item-related due to the isolated occurrence in a single animal. Salivation was observed (slight to moderate on one occasion each) for 4 females dosed at 250 mg/kg bw/day between GD 8 and 9, was considered likely secondary to gavage needle withdrawal of the high concentration dose solution, and not considered test item-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
There were two deaths at the high dose (250 mg/kg bw/day) considered related to the test item. Animal 4509 died following blood collection procedures prior to cesarean section on GD21 and Animal 4521 was found dead prior to caesarean section on GD 21. Given the proximity to blood collection for 4509, the death may have been exacerbated by stress related to the blood collection procedure; however, these were considered test item-related due to absence of similar effects at lower doses or in controls. Macroscopic changes noted in these two preterminal animals were comparable to those observed in main scheduled animals given 250 mg/kg bw/day and included pale discoloration of the liver or presence of dark foci in the stomach.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The administration of the test item resulted in a reduction of body weight and body weight change for dams dosed at 250 mg/kg bw/day from the first day of dosing and throughout the dosing period to GD 21, that became statistically significant (p ≤ 0.05, p ≤ 0.01 or p ≤ 0.001) on GD 21 (termination). Corrected body weight (body weight – uterus weight) and corrected body weight changes were also statistically significantly (p ≤ 0.05 or p ≤ 0.001) reduced at 250 mg/kg bw/day following dosing in comparison to the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a test item related reduction of food intake for dams dosed at 250 mg/kg bw/day from the first day of dosing to GD 21, that became statistically significant (p ≤ 0.05) on GD 21.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Administration of the test item by oral gavage to Sprague-Dawley rats from Gestation Days (GD) 5 to 20 resulted in pale discoloration or pale area, enlargement and/or prominent lobular architecture in the liver of 6 out 24 female rats. In addition, there were test item-related changes in spleen (small), pancreas (gelatinous) and/or stomach (dark foci or discoloration) in a few females given 250 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
There were no test item-related effects on the number of corpora lutea, implantation sites, live fetuses, sex ratio, resorptions or pre- and post-implantation losses. The gravid uterus weight for dams at 250 mg/kg bw/day was reduced in comparison to controls due to significantly lower fetal weights.

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a statistically significant test item-related effect on male, female and total (combined) fetal weights (p ≤ 0.001) at 250 mg/kg bw/day. These significant changes correlated with the reduction of gravid uterus weights at this dose level.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There was a statistically significant test item-related effect on male, female and total (combined) fetal weights (p ≤ 0.001) at 250 mg/kg bw/day. These significant changes correlated with the reduction of gravid uterus weights at this dose level.
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
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no major external or internal malformations considered related to test item administration up to 250 mg/kg bw/day. The following changes were considered background findings not uncommon in this species:
- One male fetus from the litter of dam 4503 was observed with microstomia and mandibular micrognatia (small mouth and small lower jaw).
- There were 2 fetuses from 2 litters with major external malformations. Male fetus
No. 9 from the litter of dam 3523 dosed at 50 mg/kg/day was observed with cheiloschisis (cleft lip and palatal rugae absent). The animal also had the snout was missing and small left eye (microphthalmia). The second animal was a male pup No.3 from dam 4514 dosed at 250 m/kg/day was also observed with small left eye.
- There were 2 fetuses from 2 litters with major internal malformations. Fetus No 5 from the litter of dam 3515 dosed at 50 mg/kg/day was observed with a hole in the heart from left to right ventricles and the thoracic cavity filled with clotted blood and fetus No. 8 from the litter of a dam 4516 treated at 250 mg/kg/day was observed with thoracic cavity filled with clotted blood.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of incomplete ossification of the frontal, interparietal, pubis, humerous, radius and parietal bones were statistically significantly (p≤ 0.05 to p≤ 0.001) increased for both litters and fetuses along with a statistical significant increase (p ≤ 0.001 or p ≤ 0.01) of fetuses with incomplete ossification of the femur, ilium and supraoccipital bones at 250/mg/kg bw/day were considered secondary to the effects on fetal weights/maternal toxicity. There was also a statistical significant (p ≤ 0.05 or p ≤ 0.01) increase of fetuses with incomplete ossification of the ilium and femur at 50 mg/kg bw/day that were considered to be incidental and within historical reference range.
The incidence of the control and groups treated at 15 and 50 mg/kg bw/day for the percentage of fetuses with common skeletal variants was comparable. There was a statistically significantly (p ≤ 0.001) increased values of sternebral variants (5-6) observed in fetuses at 250 mg/kg bw/day in comparison to the control that was considered to be test item related but secondary to the effects on fetal weight.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no minor internal anomalies that were related to the administration of test item up to 250 mg/kg bw/day. The following internal anomalies were considered background findings not uncommonly seen in this species:
- Fetuses No. 1 and 13 from litter 1517 (Control) were observed with left ureter dilated moderately.
- Fetus No. 9 and 11 from litter of 2508 (15 mg/kg bw/day), fetus No. 17 from litter of 3501 (50 mg/kg bw/day), fetus No. 14 from litter of dam 3503(50 mg/kg bw/day), fetus No 1 and 7 from litter of dam 3509, fetus No 4 from dam of 3512 and fetuses No. 1, 5 and 10 from litter of dam 3523 were observed with one or both ureters moderately to severe dilation. Also, at 250 mg/kg bw/day, fetus No. 4 and 6 from dam 4504; fetus No. 3, 7 and 11 from dam 4505, fetus No. 7 from dam 4511 and fetus No. 8 from dam 4519 were observed with one or both ureters dilated moderately. The renal papilla from both kidneys were small for pup No. 5 from litter of dam 3525 (50 mg/kg bw/day) and small left renal papillae from fetuses 1 and 7 from litter of dam 3509 and for fetus No 6 from litter of dam 4504 (250 mg/kg bw/day).
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed at highest dose

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Administration of test item by oral gavage daily from Gestation Days 5 to 20 (inclusive) to pregnant Sprague-Dawley rats at doses of 0, 15, 50, and 250 mg/kg bw/day resulted in maternal mortality, and reduced food consumption/body weight, body weight gain, and corrected body weight, along with gross pathological observations primarily in the liver, for dams at 250 mg/kg/day. There were also treatment-related reductions in male, female and total fetal weights and minor skeletal anomalies considered secondary to maternal toxicity generally at 250 mg/kg bw/day. Based on the results of this study, there was no embryolethality, fetotoxicity, or signs of teratogenicity at doses up to 250 mg/kg bw/day of test item when administered orally via gavage to pregnant Sprague Dawley rats.
Executive summary:

The objective of the study was to investigate the embryo-fetal developmental toxicity of the test item administered to pregnant female Sprague-Dawley rats by oral (gavage) daily from Gestation Days (GD) 5 to 20 inclusive. The test item and reference item/vehicle were administered daily, during the dosing period, as shown below:

Table 1. Dosing

Treatment

Group

Dose Level

(mg/kg bw/day)

Dose Conc.

(mg/mL)

Dose Volume

(mL/kg)

Number of Pregnant Females

1. Control*

0

0

4

24

2. Test item -Low Dose

15

3.75

24

3. Test item -Mid Dose

50

12.5

24

4. Test item -High Dose

250

62.5

24

*Group 1 animals received the reference item/vehicle (corn oil).

 

The test item was formulated as solutions in corn oil (also the control item/vehicle) and administered via oral gavage to pregnant Sprague-Dawley rats that were roughly 9-10 weeks of age, with body weights ranging from 196 to 263 grams at the start of treatment (November 1, 2015). Animals had been mated on the day prior to shipping, and on the following day, those females confirmed as mated (with vaginal plugs) were shipped. The day of receipt (day following mating) was considered Day 0 of the study. Cage side clinical observations were performed twice daily and a detailed clinical observation was performed on days of body weight recordings. Individual body weight was measured on Days 0, 3, 5, 6, 9, 12, 15, 18 and 21 of gestation. Food consumption was measured on Days 0 to 3, 3 to 5, 5 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 21 of gestation. On GD 21, the animals were euthanized by exsanguination following CO2 asphyxiation and an external macroscopic examination, including identification of all clinically-recorded lesions, as well as a detailed internal examination were performed. The reproductive tract was dissected out, the ovaries removed and corpora lutea counted. The gravid uterus was weighed. The number of implantations recorded and the pre- and postimplantations losses calculated. The uterine contents, including the placenta, were examined and the numbers of live fetuses, dead fetuses, early middle and late resorptions were recorded. Test item-related effects were noted in dams at the high dose only and included mortality in 2 of 24 animals with associated clinical signs of reduced activity, cold to touch, lying on cage floor, and decreased respiration rate, reduced food consumption/body weight, and gross changes at necropsy that included liver findings of pale discoloration/pale area, enlargement and/or prominent lobular architecture, as well as small spleen, gelatinous pancreas, and dark foci/discoloration in the stomach. There were test item-related reduced fetal body weights at the high dose; however, no effects on the number of corpora lutea, implantation sites, live fetuses, sex ratio, resorptions, or pre- and post-implantation losses. There were no incidences of major malformations, minor external or internal anomalies that were related to Neononyl Acetate at any dose levels up to 250 mg/kg/day. The incidence of incomplete ossification of the frontal, interparietal, pubis and parietal bones were significantly increased for both litters and fetuses along with a significant increase of fetuses with incomplete ossification of the supraoccipital bones at 250/mg/kg bw/day; these were considered secondary to maternal toxicity. Also noted were increase of fetuses with incomplete ossification of the ilium and femur at 50 mg/kg bw/day that was considered incidental. In summary, the administration of test item by oral gavage daily from Gestation Days 5 to 20 (inclusive) to pregnant Sprague-Dawley rats at doses of 0, 15, 50, and 250 mg/kg bw/day resulted in maternal mortality, and reduced food consumption/body weight, body weight gain, and corrected body weight, along with gross pathological observations primarily in the liver, for dams at 250 mg/kg bw/day. There were also treatment-related reductions in male, female and total fetal weights and minor skeletal anomalies considered secondary to maternal toxicity generally at 250 mg/kg bw/day. Based on the results of this study, there was no embryolethality, fetotoxicity, or signs of teratogenicity at doses up to 250 mg/kg bw/day of test item when administered orally via gavage to pregnant Sprague Dawley rats.