Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May-October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted under GLP according to OECD TG 414.

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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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., Quebec, Canada
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: 242 - 333 g
- Fasting period before study: no
- Housing: animals were housed individually in stainless steel cages.
- Diet: standard certified commercial chow (ENVIGO Certified Global Rodent Diet #2018C), ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The test item and reference item dosing formulations were prepared at least weekly. The test item was weighed and transferred into a container (or weighed directly into the container). The vehicle was then added to the final volume, followed by stirring. All formulations, including the vehicle, were stored at room temperature prior to administration.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was chosen due to its ability to solubilize the test item.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch no.: 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. The samples were stored frozen (-20 °C). An additional 15-mL sample was collected from the middle of each concentration at the start of the study for density measurement. Following confirmation of safe and intact receipt of the first set of samples, the second set of the duplicates were discarded. The analysis was performed by the analytical laboratory by gas chromatography (GC analysis) within the expected 1-month stability data range. An acceptance criterion for the formulation was ± 15% of nominal.
Details on mating procedure:
- Animals had been mated at the animal supplier on the day prior to shipping, and on the following day, those females confirmed as mated (with vaginal plugs) were shipped to the testing laboratory.
- Proof of pregnancy: vaginal plug, referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 5 - 20 of gestation
Frequency of treatment:
once daily, 7 days/week
Duration of test:
16 days; Day 5 - 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
based on oral range finding study
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
based on oral range finding study
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
based on oral range finding study
No. of animals per sex per dose:
23 P females (100 mg/kg bw/day)
24 P females (0, 300 and 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on an oral range finding gavage study conducted in rats with the test item, the high dose level selected by the Sponsor for this dose range finding study is 1000 mg/kg bw/day, with approximately 3-fold lower Mid and Low dose levels (300 and 100 mg/kg, respectively).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs (ill health, behavioral changes, etc.) and mortality were recorded on all animals twice daily throughout the course of the study. Additional clinical observations may be performed if deemed necessary to properly monitor the animals health condition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed clinical examination was performed on each animal on Gestation Days 0, 3, 5, 6, 9, 12, 15, 18 and 21 (day of necropsy).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded for all animals at least as follows: Gestation Days 0, 3, 5, 6, 9, 12, 15, 18 and 21 (prior to Caesarean section).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Individual food intake was measured on all surviving animals at least 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
- Organs examined: Reproductive tract including ovaries and gravid uterus with placenta (external macroscopic examination, including identification of all clinically-recorded lesions) as well as a detailed internal examination will be performed.
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:
Each pairwise group comparisons of interest were conducted via a two-sided test at the 5% significance level (with significant results p ≤0.001, p ≤0.01 or p ≤0.05). 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. 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 was 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 and each dose group. When the Shapiro-Wilk test on the residuals of the second ANOVA was significant or when heterogeneous group variances (p ≤ 0.05) are revealed by the Levene test, the groups were 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 group and each dose group. For each data set with more than two groups to compare, data were analyzed using the Kruskal-Wallis test. When the Kruskal Wallis test was significant (p ≤0.05), the Dunn test was used to perform the pairwise group comparisons of interest. Group incidence results are presented for each considered parameter. If there are more than two groups to compare, an overall test (including all groups) was performed using the Fisher exact test. If the overall test is significant (p ≤0.05) or if there are only two groups to compare, the pairwise group comparison of the control group with each of the other dose groups were done using Fisher’s exact test.
Indices:
- Pregnancy rate (%) = (Number of pregnant rats / Number of mated rats) x 100
- Pre-implantation loss (%) = [(Number of corpora lutea - Number of implants) / Number of corpora lutea] x 100
- Post-implantation loss (%) = [(Number of implants - Number of live fetuses) / Number of implants] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight to severe salivation in all animals at the high dose group of 1000 mg/kg bw/day beginning on GD 9 post-dose; 2/24 mid-dose animals (300 mg/kg bw/day) were observed with slight to moderate salivation once on GD 18 and GD 20.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in body weight change for females dosed at 1000 mg/kg bw/day following the first day of administration (GD 5 to 6) that was not considered to be of toxicological importance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction of food consumption was noted for females on GD 6 to 9 at 1000 mg/kg bw/day. This reduction was not considered to be toxicologically 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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL OBSERVATIONS
There was no mortality throughout the course of the study. Test-item related clinical signs of slight to severe salivation were observed in all animals dosed at the high dose of 1000 mg/kg bw/day beginning on GD 9 post-dose. 2/24 mid-dose animals (300 mg/kg bw/day) were observed with slight to moderate salivation once on GD 18 and GD 20, respectively and 1/24 control group animal was observed with severe salivation once on GD 15. Other clinical signs that included but were not limited to fur stained red or skin dry were considered incidental since they were observed in the control group or prior to the start of dosing.

BODY WEIGHT
There were no test item-related significant effects on body weight, body weight changes or corrected body weight changes up to 1000 mg/kg bw/day in comparison to the control group. There was a statistical significant (p ≤0.5 or p ≤0.01) reduction in body weight change noted for females dosed at 1000 mg/kg bw/day following the first day of administration (GD 5 to 6), that was related to the test item but not considered to be of toxicological importance as the weights rebounded on subsequent weighing. A slight reduction in corrected body weights changes was noted at 300 and 1000 mg/kg bw/day.

FOOD CONSUMPTION AND COMPOUND INTAKE
There were no test item-related effects on food consumption up to 1000 mg/kg bw/day in comparison to the control group. A statistical significant reduction of food consumption was noted for females on GD 6 to 9 at 1000 mg/kg bw/day. This reduction was not considered to be toxicologically significant as animal’s consumption were comparable or increased throughout the pregnancy.

POST-MORTEM EXAMINATIONS
There was no macroscopic tissue changes considered related to the administration of the test item.

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:
effects observed, non-treatment-related
Description (incidence and severity):
Although all animals on study were observed as having vaginal plugs on Day 0, at the end of the study, one female of the low-dose group (100 mg/kg bw/day) was not pregnant.
Other effects:
not examined
Details on maternal toxic effects:
MATERNAL PERFORMANCE
Although all animals on study were observed as having vaginal plugs on Day 0, at the end of the study, one female of the low-dose group (100 mg/kg bw/day) was not pregnant. The pregnancy rates were 100, 95.8, 100 and 100% for the control, low-dose, mid-dose and high-dose groups, respectively.

UTERINE FINDINGS
There were no test item-related effects on gravid uterus weights, the number of corpora lutea, implantation sites, live fetuses, sex ratio, dead fetuses, resorptions or pre- and post-implantation losses.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
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:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
FETAL WEIGHTS
There were no significant test item-related effects on male, female and total (combined) fetal weights up to 1000 mg/kg bw/day.

EXTERNAL AND INTERNAL MALFORMATIONS
There were no major malformations or minor external, internal or skeletal anomalies that were considered related to treatment with the test substance at any dose level up to 1000 mg/kg bw/day.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The administration of the test substance by oral gavage daily from Gestation Days 5 to 20 inclusive to gravid female Sprague-Dawley rats at dose levels of 0, 100, 300, and 1000 mg/kg bw/day was well tolerated. Clinical signs such as salivation and reduction of food consumption and body weights at the high-dose group (1000 mg/kg bw/day) were recorded. However, there was no evidence of embryolethality, fetotoxicity or teratogenicity at doses up to 1000 mg/kg/day. Thus, the NOAEL for maternal and developmental toxicity was considered to be greater than 1000 mg/kg bw/day.