Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 JAN 2018 to 25 MAY 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
Screening study for reproductive toxicity accordinf to OECD TG 421
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 18 JAN 2018 to 25 MAY 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD TG 421
Version / remarks:
version from 29th July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: Milli-Q water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to specific group of rats at approximately the same time each day (varying by ± 3 hours), for a period of 35 days (which includes two weeks prior to mating, during the mating period and post mating) after which they were sacrificed afterovernight fasting.
Females: The dose formulation was administered orally by gavage to the specific group of rats at approximately the same time each day (varying by ± 3 hours), two weeks prior to the mating period and was continued through mating, pregnancy and up to LD 13 (Day 43-57). On LD 14, the females were sacrificed after overnight fasting.
The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle only (for males 35 days and for females 43-56 days). The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat. Similarly, vehicle was administered to rats in the vehicle control group at an equivolume of 10 mL/kg body weight.
Frequency of treatment:
Daily
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed at any of the doses tested in both sexes.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption was unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in the haematological parameters analysed across the groups in both male and female rats at the end of treatment period.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in adult males (at termination), adult females (on lactation Day 13) and pups (on lactation Days 4 and 13) across the treatment groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in the terminal fasting body weights, organ weight and their ratios in adult male and female rats.
In adult male rats, increased relative weight of testes (11%) at 60 mg/kg Bwt/day and adrenal weight (14%) at 120 mg/kg Bwt/day were considered incidental in nature due to lack of dose progression and/or microscopic correlation.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related adverse gross pathological changes observed in reproductive organsin both males and females rats.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related histopathological changes noted (including reproductive tissues) in any of the adult male and female rats. The qualitative assessment of stages of spermatogenesis in testes did not reveal any test item associated findings in adult rats.

Few randomly distributed microscopic findings observed in some of the rats were considered as incidental background findings commonly noted in this age group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in adult males (at termination) and adult females (on lactation Day 13) across the treatment groups.
Dose descriptor:
NOAEL
Effect level:
>= 120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no

The summary tables of main results are attached as background materials under “Overall remarks, attachments”.

Conclusions:
To summarize, daily oral (gavage) administration of test item to Wistar rats at the dose levels 30, 60 and 120 mg/kg Bwt/day for 2 weeks prior to mating, during mating, post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females at all the tested doses had no effects on general health, body weights, food consumption. There were no adverse effects on haematology parameters, thyroid hormone profile (TSH and T4), terminal body weight, organ weight and their ratios, gross pathology and histopathology in all animals at all dose levels tested.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
version from 29th July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Water added up to 100 %.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana

- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 11-13 weeks
- Weight at study initiation: Males: 266.59 to 344.47 g; Females: 193.44 to 234.17 g;

- Housing: Pre-mating: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes.
Mating and post-mating:
During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (GD 20).
Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects during pre-mating period and post-mating period for males and pre-mating period for females. Enrichment was changed along with cage once a week.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Envigo, P.O. Box 44220, Madison, WI 53744-4220, was provided ad libitum to animals. A sample of the diet was retained and discarded.

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: Start: 30 December 2017 End: 03 January 2018

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21−24°C
- Humidity (%): 59 – 68 %
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
IN-LIFE DATES: From: 04 January 2018 To: 12 April 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Milli-Q water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared fresh daily before dosing and was administered to the respective group animals.
The required quantity of test item was weighed in pre-calibrated beaker*. A small volume of vehicle (Milli-Q® water) was added mixed well using glass rod. The final volume was made up with the vehicle to get the required final concentration.
Pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated measuring cylinder to the final volume of 200 mL. The measured Milli-Q® water was transferred into a clean beaker and upper and lower meniscus of Milli-Q® water was marked on the beaker using a glass marker. After marking these lines, the Milli-Q® water was discarded and the beaker was allowed to dry. The upper meniscus was used to make up the volume.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Milli-Q® water was used as vehicle for dose formulation preparation as the same vehicle was used in the dose range finding study for Reproduction/Developmental Toxicity Screening Test in Wistar Rats (study no. N3484). Further, same vehicle was used in authenticity, method validation and stability.
Details on mating procedure:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: 21 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged (how): One per cage
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd month (Day 34) of the treatment period and were analysed in-house. For each set, composite samples were drawn in three replications and in case of control, duplicate composite samples were drawn
The analysis was done as per the method validated under Eurofins Advinus Study No.: G14942. One set of samples were analysed for concentration (a.i) analysis.
Dose formulations were considered acceptable as the overall mean results were within ± 20% of the theoretical concentration and the overall relative standard deviation (RSD) was less than 20%.
The unused back up samples were disposed as analysis results of the first set of samples were within the acceptable limits.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to specific group of rats at approximately the same time each day (varying by ± 3 hours), for a period of 35 days (which includes two weeks prior to mating, during the mating period and post mating) after which they were sacrificed after overnight fasting.
Females: The dose formulation was administered orally by gavage to the specific group of rats at approximately the same time each day (varying by ± 3 hours), two weeks prior to the mating period and was continued through mating, pregnancy and up to LD 13 (Day 43-57). On LD 14, the females were sacrificed after overnight fasting.
The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle only (for males 35 days and for females 43-56 days).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control group at an equivolume of 10 mL/kg body weight.
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: [15 to 16] weeks
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels of 30 (G2), 60 (G3) and 120 (G4) mg/kg/day were selected for this study in consultation with sponsor based on the outcome of dose range finding study for Reproduction/Developmental Toxicity Screening Test in Wistar Rats (study no. N3484). Considering the purity of 15.4% (active content) as per COA generated by Eurofins Advinus Test facility (AUTH/891), these dose levels corresponds to approximately 194.8 (G2), 389.6 (G3) and 779.2 (G4) mg/kg of the test item.
In addition to the test doses, vehicle control group was included. Animals in the vehicle control group were handled in a manner similar to the treatment groups except for test item administration.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Oestrous cyclicity (parental animals):
Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [Yes]
- If yes, maximum of [8] pups/litter ([4]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other:] Yes

GROSS EXAMINATION OF DEAD PUPS:
[Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] Yes

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in [Section 9.9.2 in Main report] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] Yes

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in [Section 9.9.2 in Main report] were prepared for microscopic examination and weighed, respectively.
Statistics:
Statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Version 12.0. All data of quantitative variables like body weight, food consumption, oestrous cycle length, hormone levels, ano-genital distance, ano-genital index and organ weights and organ weight ratios data were tested for homogeneity of variances (Levene’s test) within the group before performing One-Way Analysis of Variance (ANOVA). When the data found to be non-optimal (non-normal or heteroschedastic), ANOVA was done using suitable transformation. Comparison of means between treatment groups and vehicle control group was done using Dunnett’s test when the overall treatment ‘F’ test is found significant.
Post implantation loss (%), no. of implantations, pre-coital interval (days), mean litter size, sex ratio and gestation length (days) were analysed after suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was performed for testing the differences found significant.
Z test was performed for testing the differences in proportions for mating, fertility and survival indices.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the tests were designated throughout the report as stated below:
+/-: Significantly higher (+)/lower (-) than the vehicle control group
Reproductive indices:
a. Male mating index (%) = (Number of males with evidence of mating / Number of males cohabited) x
100
b. Male fertility index (%) = (Number of males siring a litter / Number of males cohabited) x 100
c. Female mating index (%) = (Number of females mated / Number of females cohabited) x 100
d. Female fertility index (%) = [Number of pregnant females (confirmed at necropsy) / Number of
females used for mating] x 100
e. Mean number of implantations/group = (Total number of implantations / Total number of pregnant
animals)
f. Post implantation loss (%) = (Number of implantations - Number of live pups / Number of
implantations) x 100
Offspring viability indices:
a. Mean litter size per group = (Total Number of pups / Total Number of littered animals)
b. Mean viable litter size = (No. of viable pups on Day 1 / No. of females littered)
c. Live birth index (%) = (No. of viable pups born (at first observation)) / (Total no. of pups born (at first observation)) x 100
d. Day 4 survival index (%) = (Number of viable pups on lactation Day 4 / Number of viable pups born) x 100
e. Sex Ratio (%) = (No. of male pups born / Total No. of pups born) x 100
f. Ano-genital Distance Ratio (mm/g1/3) = (Ano-genital distance / Cube root of body weight)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed at any of the doses tested in both sexes.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality were observed in any of the tested doses.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption was unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in the haematological parameters analysed across the groups in both male and female rats at the end of treatment period.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in adult males (at termination), adult females (on lactation Day 13) and pups (on lactation Days 4 and 13) across the treatment groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in the terminal fasting body weights, organ weight and their ratios in adult male and female rats.

In adult male rats, increased relative weight of testes (11%) at 60 mg/kg Bwt/day and adrenal weight (14%) at 120 mg/kg Bwt/day were considered incidental in nature due to lack of dose progression and/or microscopic correlation.

There were no significant changes in terminal body weights and thyroid weights in male/female pups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related adverse gross pathological changes observed in reproductive organs in both males and females rats.

Gross pathological examination of dead pups and pups sacrificed on LD 13 did not reveal any abnormalities at all the dose levels tested.

The cause of infertility in the female rats (G1: Ru5480; G2: Ru5491, Ru5498; G3: Ru5518 and G4: Ru5535, Ru5536) could not be ascertained as no corresponding gross and microscopic changes were observed in reproductive organs.

Single incidence of unilateral absent Cowper’s gland (G1M: Ru5467), enlarged spleen (G3F: Ru5514) and dilated uterus (G4F: Ru5536) were considered as incidental finding and not related to test item administration
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related histopathological changes noted (including reproductive tissues) in any of the adult male and female rats. The qualitative assessment of stages of spermatogenesis in testes did not reveal any test item associated findings in adult rats.
There were no test item-related microscopic changes in thyroid gland of pups of all the dose levels.
Few randomly distributed microscopic findings observed in some of the rats were considered as incidental background findings commonly noted in this age group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in adult males (at termination) and adult females (on lactation Day 13) across the treatment groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during treatment period (prior to cohabitation).
The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
The calculated mean oestrous cycle length was 4.11, 4.08, 4.05 and 4.09 days in vehicle control, 30, 60 and 120 mg/kg Bwt/day doses, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.
Pre-coital time was determined (in days) from oestrous cycle evaluation from the day of cohabitation to the evidence of mating.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The qualitative assessment of stages of spermatogenesis in testes did not reveal any test item associated findings in adult rats.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups.

Details on results (P0)

To summarize, daily oral (gavage) administration of test item to Wistar rats at the dose levels 30, 60 and 120 mg/kg Bwt/day for 2 weeks prior to mating, during mating, post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females at all the tested doses had no effects on general health, body weights, food consumption, pre-coital time, gestation length, mating and fertility and survival indices. There were no adverse effects on haematology parameters, thyroid hormone profile (TSH and T4), terminal body weight, organ weight and their ratios, gross pathology and histopathology in adult animals and pups at all dose levels tested.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The mean numbers (and total number) of male and female pups per litter were not affected by the treatment at all the doses tested.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The weight of male and female pups perlitter were not affected by the treatment at all the doses tested.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
no effects observed
Description (incidence and severity):
No changes attributable to test item were detected in the Ano-genital distance (AGD), body weight and ratio of AGD to the cube root of body weight of either sex at all the tested doses.
The male pups did not exhibit areolae/nipple retention on PND 13 at any of the doses tested.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no significant changes in terminal thyroid weights in male/female pups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathological examination of dead pups and pups sacrificed on LD 13 did not reveal any abnormalities at all the dose levels tested.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic changes in thyroid gland of pups of all the dose levels.
Other effects:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in pups (on lactation Days 4 and 13) across the treatment groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F2 generation

General toxicity (F2)

Dermal irritation (if dermal study):
not examined

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

The summary tables of main results are attached as background materials under “Overall remarks, attachments”.

Applicant's summary and conclusion

Conclusions:
To summarize, daily oral (gavage) administration of test item to Wistar rats at the dose levels 30, 60 and 120 mg/kg Bwt/day for 2 weeks prior to mating, during mating, post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females at all the tested doses had no effects on general health, body weights, food consumption, pre-coital time, gestation length, mating and fertility and survival indices. There were no adverse effects on haematology parameters, thyroid hormone profile (TSH and T4), terminal body weight, organ weight and their ratios, gross pathology and histopathology in adult animals and pups at all dose levels tested.

No Observed Adverse Effect Level
Daily oral (gavage) administration of test substance to male and female Wistar rats at dose levels of 30, 60 and 120 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters.
The No Observed Adverse Effect Level (NOAEL) of the test substance for reproductive and developmental toxicity is considered to be 120 mg/kg Bwt/day.
Executive summary:

The purpose of this study in Wistar Rats was to generate limited information concerning the effects of test substance on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was weighed and dissolved in Milli-Q®water and administered to rats at the graduated dose levels of 30, 60 and 120 mg/kg/day for low dose (G2), mid dose (G3), and high dose (G4) group rats, respectively. The rats in the vehicle control group (G1) received vehicle (Milli-Q®water) alone. The dose volume administered was 10 mL/kg body weight. Each group in the experiment was comprised of 10 males and 10 female rats. The prepared dose formulations were administered once daily to specific groups of rats prior to mating, during mating and post-mating periods for males, prior to mating, during conception and pregnancy and after delivery up to Lactation Day (LD) 13 for females.

The identity of the test substance was provided by the Sponsor by a Certificate of Analysis (CoA). To confirm the purity, the test item was authenticated at the Test facility. The test item was also re-authenticated at the end of this study. The stability of test item in the vehicle was carried out separately under Eurofins AdvinusStudy No. G14942 at 2.5 and 50 mg/mL concentrations. Based on the results, the test item was found to be stable for 4 days when stored at room temperature.The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2ndmonth (Day 34) of the treatment period. The results indicated that the analysed concentrations were within ± 20% of variations from the nominal concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly during pre-mating period. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7 and 13. The number, weight, survival and mortality of pups were recorded during the lactation period. The ano-genital distance of each pup was measured on LD 0. All survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Prior to necropsy, blood samples were collected for haematology andthyroid hormone analysis from males and females (LD 13) and for thyroid hormone analysis from available pups on LDs 4 and 13. The animals were subjected to detailed necropsy at sacrifice and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid glands from available one male and one female pup from each litter were collected for histopathological examination.

Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. Histopathological examination of the testes were also includes a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups. The reproductive organs of all not littered females (G1: Ru5480; G2: Ru5491, Ru5498; G3: Ru5518 and G4: Ru5535, Ru5536) were examined in all the dose groups

The available thyroid gland from a male and a female pup per litter (randomly selected) were also evaluated from all the groups

Under the experimental conditions employed, the following results were obtained:

·        There were no treatment-related clinical signs or mortality observed at any of the doses tested in both sexes.

·        The mean body weight in males and pre-mating period females were unaffected by the treatment at all the doses tested.

·        The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the doses tested.

·        Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, fertility indices of sires and dams and survival indices at all the tested doses.

·        Mean number and weight of male, female and combined sex pups in treated groups were comparable to control group.

·        There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups.

·        No treatment-related changes were observed in the ano-genital distance and ratio to ano-genital distance tocube root of body weightat any of the doses tested when compared to the vehicle control group.

·        No areola/nipple retention was observed in male pups on PND 13 at any of the doses tested.


·        gross pathology and histopathology in adult animals and pups at all dose levels tested.

No Observed Adverse Effect Level

Daily oral (gavage) administration of test substance to male and female Wistar rats at dose levels of 30, 60 and 120 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters. The No Observed Adverse Effect Level (NOAEL) of the test substance for reproductive and developmental toxicity is considered to be 120 mg/kg Bwt/day.