Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-12 weeks.
- Weight at study initiation: All animals were within +/- 20% of the sex mean.
- Housing: Males and females were housed in groups of 5 in macrolon cages. During mating, males and females were caged together in macrolon cages. During lactation, pups were kept with the dams until termination.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet, except during motor activity assessments for the selected males and females, and for 24 hours prior to blood chemistry sampling.
- Water (e.g. ad libitum): Free access to tap water, except during motor activity assessments for the selected males and females.
- Acclimation period: At least 5 days prior to the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): 10 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance. Test formulations were stored at room temperature protected from light until use. Thevehicle was chosen based on the results of trial formulations performed prior to treatment.

VEHICLE
- Concentration in vehicle: 0, 2.5, 5 and 10 mg/mL
- Amount of vehicle (if gavage): Dose volume of 5 mL/kg/day
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of mating was apparent.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 post-coitum.
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was conducted at a single sample point during the study according to a validated method. Samples of formulation were analysed for homogeniety (highest and lowest concentration) and accuracy of preparation (all concentrations). The stability in the vehicle over 6 hours at room temperature under protection from light was also determined (highest and lowest concentration). The accuracy, homogeniety and stability of test samples as appropriate were reported to be in the satisfactory ranges, ensuring the acceptability of dosing formulations.
Duration of treatment / exposure:
Males were exposed for 29 days (i.e. 2 weeks prior to mating, during mating and upto the day prior to scheduled necropsy). Females were exposed from 42-55 days (i.e. during 2 weeks prior to mating, during mating, during post-coitum (gestation) and during at least 4 days of lactation up to the day prior to scheduled necropsy). The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Frequency of treatment:
Parental males and females once daily via oral gavage
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 12.5, 25 and 50 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
- A total of 80 rats were used on study (40 males and 40 females).
- Each test group consisted of 10 males and 10 females (including 10 males and females in the control groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on the results of a 10-day dose range finding study.

Examinations

Parental animals: Observations and examinations:
See Chapter 7.5.1, record Repeated dose toxicity: oral_(OECD 422)_Key_WIL_2015
Estrous cyclicity (parental animals):
Determination of estrous cycling was not conducted as is not an explicit guideline requirement.
Sperm parameters (parental animals):
Not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
The numbers of live and dead pups (Each day of lactation; defects and causes of death were evaluated), daily clinical signs, bodyweights (Days 1 and 4) and sex (Days 1 and 4). Using this information the percentage of live males and females at first litter check was calculated, the percentage of postnatal loss and the viability index.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities.
Postmortem examinations (parental animals):
Necropsy and histopathology procedures are described in Chapter 7.5.1, record Repeated dose toxicity: oral_(OECD 422)_Key_WIL_2015
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring surviving to planned terminated were killed by decapitation on Days 5-7 of lactation.
- These animals were subjected to postmortem macroscopic examination (external anomalies). The stomach of pups not surviving to scheduled termination were examined for the presence of milk if possible.

GROSS NECROPSY
- Gross necropsy consisted of a check for external anomalies, including that in the oral cavity.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means
and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet different test statistics values.
Reproductive indices:
Mating index (%), fertility index (%), conception index (%), gestation index (%), duration of gestation (days) were the determined reproductive indices.
Offspring viability indices:
Percentage live males/females at first litter check (%), percentage of postnatal loss, and viability index were the determined offspring viability indices.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
not examined
Description (incidence and severity):
Examined in section (7.5.1)
Body weight and weight changes:
not examined
Description (incidence and severity):
Examined in section (7.5.1)
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Examined in section (7.5.1)
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
Examined in section (7.5.1)
Gross pathological findings:
not examined
Description (incidence and severity):
Examined in section (7.5.1)
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Examined in section (7.5.1)
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Reproductive performance
There were no treatment-related differences in:
- Mating indices
- Fertility indices
- Conception indices
- Pre-coital time
- Number of corpora lutea and implantation sites

One female receiving 50 mg/kg/day, did not show evidence of mating. This was considered to be within the normal range for animals of this age and strain.

Selected results by dose level (0, 12.5, 25 or 50 mg/kg bw/day).
- females mated: 10/10, 10/10, 10/10, 9/10
- females pregnant: 9/10, 9/10, 10/10, 8/10
- females with live pups: 9/10, 9/10, 10/10, 8/10
- Mating index (%): 100, 100, 100, 90
- Fertility Index (%): 90, 90, 100, 80
- Conception Index (%): 90, 90, 100, 89
- Gestation Index (%): 100, 100, 100, 100

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproductive parameters were observed up to the highest dose level of 50 mg/kg.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Developmental parameters considered unaffected by treatment are sex ratio, clinical signs, external macroscopy, gestation and parturition and maternal care.

BODY WEIGHT (OFFSPRING)
At 25 and 50 mg/kg, mean pup bodyweights of both male and female pups statistically significantly slower on day 4 of lactation. Bodweight gain over days 1-4 based on combined weights for both sexes was approximately 15 and 20% lower than control mean bodyweight gain at 25 and 50 mg/kg, respectively. There were no statistically significant differences in pup bodyweight on day 1 of lactation.

GROSS PATHOLOGY (OFFSPRING)
No external anomalies were observed in any offspring, and no mortality occured that was considered related to treatment.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
12.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 25 and 50 mg/kg mean pup bodyweight gain of pups of both sexes was lower than in controls. Given the magnitude of lower pup bodyweight gain, this was considered to be an adverse developmental effect. No findings were observed at 12.5 mg/kg.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No reproductive toxicity was observed at any dose tested. With regards to developmental effects, mean pup bodyweight at 25 and 50 mg/kg on Day 4 of lactation was reduced compared with controls, and given the magnitude of the lower pup bodyweight gain, was considered to be adverse. This finding is more likely attributed to parental toxicity, as the lowest mean pup bodyweight gain per litter was noted for dams that showed weight loss during lactation. On Day 1 of lactation there was also no statistically significant differences in pup bodyweights at all doses compared with controls. As such the NOAEL for reproduction and developmental effects was considered to be at least 50 mg/kg and 12.5 mg/kg respectively.