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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Product number M2933; Batch no. SLBN0344V
- Expiration date of the lot/batch: 30.04.2020
- Purity: 100.2% (Titr. by NaOH, anhyd.)
- Water content: 7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (RT)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle:soluble in vehicle up to the highest concentration (200 mg/mL). Test item proved to be stable in the vehicle at RT for at least 4 days (preparation period)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain: Hsd.Han (of Wistar origin)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., 1103 Budapest, Cserkesz u. 90
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks (both sexes)
- Weight at study initiation:
males: 327-381
females: 202-244g
- Fasting period before study: no
- Housing: before mating: 2 animals of the same sex/cage; mating: 1 male and 1 female/cage; pregnant females: individually; males after mating: 2/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY:
food: ssniff SM R/M-Z+H complete diet, changed weekly; tap water, fresh every day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3°c
- Humidity (%): 30-70%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was solved in distilled water in concentrations of 20, 60 and 200 mg/mL. Dosing solutions were prepared not longer than for four days before the administration and stored at room temperature until use.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Batch no. of Aqua purificata (distilled water):
1702-5502, 1702-5510, 1703-5503 (supplier: Parma Produkt Kft., Budapest, Hungary)

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation or 14 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): individually
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations was performed by LC/MS/MS in the Analytical Laboratory of Test Facility twice during the study. Recoveries of the test item in the dosing formulations ranged from 100 - 105% of the nominal concentrations.
Stability and homogeneity in distilled water over the range of relevant concentration has been demostrated at room temperature for at least four days (recoveries from 98% to 105%), i.e. the maximum period of solutions administered.
Duration of treatment / exposure:
females: 56-66 days (depending on mating effectiveness)
males: 56-57 days (depending on mating effectiveness)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 male and 12 female per dose and control
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results from a 14-day dose-range finding experiment that used the same concentrations
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): random
Positive control:
none

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 (starting on Day 0)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes (body weight gain only)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable

OTHER: none

Oestrous cyclicity (parental animals):
The estrous cycle was monitored by examining vaginal smears each day before treatment start for each rat being considered for the study for two weeks. The estrous cycle was evaluated and considered for randomisation. However, some animal hot showing typical 4-5 day cycles were included in the study due to the large amount of rats with irregular cycles.
Vaginal smears were prepared and estrous cycle was monitored daily from the beginning of the treatment period (2 weeks pre-mating period) and during the mating period until evidence of mating.
Vaginal smears were also prepared on the day of necropsy.
Smears were stained (1% aqueous methylene blue solution) and examined microscopically.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- testis weight
- epididymis (and prostate and seminal vesicles with coagulating glands) weight
- histopathology of stages of spermatogenesis in the male gonads
- histopathology of interstitial testicular cell structure

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups
- From at least 2 pups per litter, blood samples for T4 analysis were collected at post-natal day 4 and post-natal day 13.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on day 55
- Maternal animals: All surviving animals 14 days post-partum

GROSS PATHOLOGY: Yes
- adrenal glands
- bone with bone marrow and joint (femur)
- brain (representative regions: cerebrum, cerebellum, pons and medulla oblongata)
- eyes (lachrymal gland with Harderian glands)
- female mammary gland
- gonads (testis with epididymides, ovaries, uterus with fallopian tube and vagina)
- gross lesions
- heart
- kidneys
- large intestines (caecum, colon, rectum, incl. Peyer's patches)
- liver
- lungs (with main stem bronchi)
- lymph nodes (submandibular, mesenteric)
- muscle (quadriceps)
- esophagus
- pancreas
- pituitary
- prostate
- salivary glands (submandibular)
- sciatic nerve
- seminal vesicle with coagulating gland
- skin
- small intestines (representative regions: duodenum, ileum, jejunum)
- spinal cord (at three levels: cervical, mid-thoracic and lumber
- spleen
- sternum
- stomach
- thymus
- thyroid + parathyroid
- trachea
- urinary bladder

HISTOPATHOLOGY : Yes (control and high dose group)
Histopathological examinations were performed on the following preserved organs and tissues of ALL animals in the control and high dose groups (12 per sex):
- Ovaries (Primodial, secondary and teriary follicles; Corpora lutea)
- Uterus
- Vagina
- Testes
- Epididymides
- Seminal vesicle with coagulating gland
- thymus (one male in low dose group due to hemorrhage)
The tissues indicated above were prepared for microscopic examination and weighed, respectively.
In particular, stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure were examined.

Full histopathological examinations were performed on the following preserved organs and tissues of selected animals in the control and high dose groups (5 per sex):
- Adrenal glands
- Aorta
- Bone marrow
- Brain
- Cecum
- Colon
- Duodenum
- Eyes + optic nerves
- Esophagus
- Harderian glands
- Heart
- Ileum
- Jejunum
- Kidneys
- Lachrymal glands
- Liver
- Lungs
- Mesenteric lymph nodes
- Muscle (quadriceps)
- Pancreas
- Pituitary
- Prostate
- Rectum
- Salivary glands (subm.)
- Sciatic nerve
- Skin
- Spinal cord
- Spleen
- Sternum
- Stomach - forestomach
- Subm. lymph nodes
- Thymus
- Thyroid + parathyroid
- Trachea
- Urinary bladder
From all parent rats, blood samples for T4 analysis were collected at termination males) or on post-partum day 13 (females).
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed latest at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY: Yes, but no detailed information available.

HISTOPATHOLOGY / ORGAN WEIGTHS: none
Statistics:
Depending on variance homogeneity between groups (Bartlett's test), parametric or non-parametric (Kruskal-Wallis test) ANOVA was performed, with subsequent inter-group comparisons (Duncan multiple range test or Mann-Whitney U-Test) in case of significant ANOVA results. If applicable, the Chi-square test was performed.
Reproductive indices:
Male reproduction data:
- Male mating index
- Male fertility index

Female reproduction and delivery data:
- Female mating index
- Female fertility index
- Gestation index
Offspring viability indices:
- Post implantation mortality
- post-natal mortality
- survival index
- sex ratio

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Pilorection, decreased body tone and paleness was observed in one female of the control group on the day of delivery and on lactation day 0. No clinical signs were observed in any other rat.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in any group during the course of study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weight and body weight gain between the control and the three treated groups were observed. On female in the high dose group was not pregnant, so that its body weight was evaluated in the pre-mating period only.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some sporadic statistically differences in some parameters were not related to dose and considered of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Kidney weights (absolute and relative) were present in a single male rat in the mid dose group. In the absence of corresponding histopathological findings and a dose relation, this was considered to be not toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Findings were only present in single animals (of both sexes) in most groups including control. In the absence of corresponding histopathological findings, these were considered to be not toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no significant differences between the control and the treated groups in the number or percentage of rats with regular cycles, mean length of cycles, mean number of days in estrous or diestrous during the pre-mating period. The mean number of days in proestrous was statistically significantly higher in the 300 mg/kg bw dose group (compared to control). As all females in this group had normal and regular estrous cycles during the pre-mating period and no dose-response was observed, this difference was considered to be unrelated to the test substance.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group one pair failed to mate during the 14-day mating period, resulting in a lower copulatory index as compared to the treatment groups.
A statistically significant lower fertilty index was observed in the male 1000 mg/kg bw day group as compared to control, because one male mated, but the female was not pregnant. As the fertility index was within the historical controls, this effect was considered to be not treatment related. This non-pregnancy also resulted in a lower female fertility index, which also was within the historical controls and thus not considered to be treatment related.
The gestation index in the mid-dose treatment group (300 mg/kg bw/day) was significantly lower as compared to control, as one female (of 12) did not deliver live pups. As this effect was not dose-dependent, it was considered to be indicative of biological variation and not treatment related.

Numbers of pregnant females and dams delivered, the mean number of implantations, post-implantation loss, duration of pregnancy and pups births (total, live, still, viable) and live birth index were similar in all groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
The highest mean number of dead offspring per litter between post-natal days 0 and 13 was observed in the control and the 300 mg/kg bw/day groups (1.3 dead pups per litter) while in the 100 and 1000 mg/kg bw/day groups it was only 0.2 per litter. The survival indices are in good correlation with the mean number of dead pups. These differences did not attain statistical significance and there is no dose dependence observed and, therefore, they are considered to have no toxicological relevance.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A test item related effect on the body weight development of the offspring was not found.
The mean litter weights as well as the mean litter weight gain were similar in the control and in all test item treated groups (100, 300 and 1000 mg/kg bw/day) on postnatal days 0, 4 and 13.
Slight changes with statistical significance were detected in the mean pup weights and mean pup weight gain; however, these minor differences without a relation to dose are considered to have no toxicological relevance.
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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
Histopathological findings:
not examined

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:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
results refer to F1 offsprings

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)

Any other information on results incl. tables

The reproductive/developmental toxicity of the test substance was screened in a experimental study according to OECD test guideline 422 under GLP-conditions. The substance, solved in distilled water, was administered to Wistar rats of both sexes by oral gavage in doses of 100, 300 and 1000 mg/kg bw daily for 56 to 66 days, starting two weeks before mating and ending a day before necropsy, i.e females' lactation days 12 -20. Observations included mortality, clinical signs, body weight, food consumption, estrous cycle (two weeks before treatment until evidence of mating), mating, pregnancy and delivery process, as well as development of offsprings. Litters were weighed and offsprings observed until post-natal day 13. All parent animals were subjected to gross pathology, specifically measuring weight of male reproductive organs. Histopathology was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland (in control and high groups). The substance did not adversely affect the reproductive performance in parental males and females. The development of the F1 offsprings was not impaired. Based on these results, the NOAEL for the reproductive performance in both sexes and for the development of F1 offsprings was 1000 mg/kg bw.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, the test substance did not cause signs of systemic toxicity in parental male and female Hsd.Han: Wistar rats at 100, 300 or 1000 mg/kg bw/day doses administered by oral gavage and the test item did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female rats at the doses of 100, 300 or 1000 mg/kg bw/day.
The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Executive summary:

The reproductive/developmental toxicity of the test substance was screened in a experimental study according to OECD test guideline 422 under GLP-conditions. The substance, solved in distilled water, was administered to Wistar rats of both sexes by oral gavage in doses of 100, 300 and 1000 mg/kg bw daily for 56 to 66 days, starting two weeks before mating and ending a day before necropsy, i.e females' lactation days 12 -20. Observations included mortality, clinical signs, body weight, food consumption, estrous cycle (two weeks before treatment until evidence of mating), mating, pregnancy and delivery process, as well as development of offsprings. Litters were weighed and offsprings observed until post-natal day 13. All parent animals were subjected to gross pathology, specifically measuring weight of male reproductive organs. Histopathology was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland (in control and high groups). The substance did not adversely affect the reproductive performance in parental males and females. The development of the F1 offsprings was not impaired. Based on these results, the NOAEL for the reproductive performance in both sexes and for the development of F1 offsprings was 1000 mg/kg bw.