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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
Study period:
08 April - 16 July 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

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

Test material

Constituent 1
Test material form:
liquid
Remarks:
Pale yellow liquid
Specific details on test material used for the study:
Storage conditions: At ambient temperature (15 to 25ºC) in the dark, under nitrogen.
No correction factor was applied for the purity.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK)
- Females nulliparous and non-pregnant: yes (estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the study)
- Age at study initiation: Males 84 to 90 days old, females 98 to 104 days old.
- Weight at study initiation: Males 307 to 350 g, females 195 to 250 g.
- Fasting period before study: No
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily. Number of animals per cage: Pre-pairing up to four animals of one sex, pairing one male and one female, males after mating up to five animals, during gestation one female, during lactation one female + litter. Environmental enrichment was supplied.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply, ad libitum
No specific contaminant was known that may have interfered with or prejudiced the outcome
of the study and, therefore, no special assay was performed.
- Acclimation period: Males six days before commencement of treatment, females 20 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS (SET CONDITIONS)
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 April 2020 To: 20 June 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a suitable container, then vehicle was added gradually while manually stirring with a spatula/rod until a homogeneous solution was obtained. Vehicle was then added to make the required volume. Following completion all containers had the head space purged with nitrogen. All formulations were prepared using glass containers.
Formulations were prepared weekly and stored refrigerated (2-8°C).

VEHICLE
- Concentration in vehicle: 20, 66 and 200 mg/mL
- Amount of vehicle: 5 mL/ kg bw
Details on mating procedure:
Pairing commenced after a minimum of two weeks of treatment.
Male/female ratio was 1:1 from within the same treatment groups.
Duration of pairing was up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the suitability of the proposed mixing procedures and the stability of the test item in dried and de-acidified corn oil formulations at concentrations of 10 and 200 mg/mL for one day at ambient temperature (15-25°C), for 15 days during refrigerated storage (2-8°C) and for 15 days following frozen storage (-15 to -25°C) were confirmed.

Samples of each formulation prepared for administration in Week 1 and the final week of treatment were analyzed for achieved concentration of the test item using a validated method.
Duration of treatment / exposure:
Males were treated daily for two weeks before pairing and throughout pairing up to necropsy for a minimum of four consecutive weeks.
Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
330 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:
10
Control animals:
yes, concurrent vehicle

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily
- A detailed physical examination was performed on each animal to monitor general health
according to the following schedule:
F0 males: Once each week
F0 females: Once each week until pairing, Gestation phase - Days 0, 7, 14 and 20, Lactation phase - Days 1, 7 and 13

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization.
Males: Before dosing on the day that treatment commenced (Week 0) and weekly thereafter and on the day of necropsy.
Females: Before dosing on the day that treatment commenced (Week 0) and weekly before pairing. Days 0, 7, 14 and 20 after mating. Days 1, 4, 7, and 13 of lactation.
On the day of necropsy.

FOOD CONSUMPTION:
- Weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 6-13 and 13-20 after mating
Days 1-4, 4-7 and 7-13 of lactation.

Thyroid hormone analysis:
Samples from offspring on Day 13 of age and F0 males were assessed for levels of thyroxine (T4).
Oestrous cyclicity (parental animals):
Dry smears: For 15 days before pairing using cotton swabs.
Wet smears: For 14 days before treatment (all females including spares);
animals that failed to exhibit 4-5 day cycles were not allocated to study. After pairing until mating (for a maximum of 14 days). For four days before scheduled termination (nominally Days 10 to 13 of lactation).
Sperm parameters (parental animals):
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.
Postmortem examinations (parental animals):
All adult animals were subject to a detailed necropsy.
Pathology procedures are listed below.
Time of Necropsy:
F0 males after four weeks of treatment.
F0 females failing to produce a viable litter Day 25 after mating.
F0 females Day 13 of lactation.
F1 offspring Day 13 of age.

For females, for each uterine horn the number of implantation sites was counted.
Postmortem examinations (offspring):
Premature deaths: Where possible fresh external macroscopic examination with an assessment of stomach for milk content. Grossly externally abnormal pups were retained.
Offspring at scheduled termination - Day 4: Externally normal offspring were discarded without examination. Externally abnormal offspring identified on dispatch to necropsy were examined externally, and retained pending possible future examination.
Offspring at scheduled termination - Day 13: All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities were retained in appropriate fixative.
Statistics:
The statistical methods used are attached below.
Reproductive indices:
Percentage mating (%) = (Number of animals mating/Animals paired) x 100;
Conception rate (%) = (Number of animals achieving pregnancy/ Animals mated) x 100;
Fertility index (%) = (Number of animals achieving pregnancy/ animals paired) x 100;
Gestation index (%) = (Number of live litters born/ Number pregnant) x 100
Offspring viability indices:
Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100;
Live birth index (%) = (Number live offspring on Day 1 after littering/ Total number of offspring born) x 100;
Viability index (%) = (Number of live offspring on Day 4 (before blood sampling)/ Number of live offspring on Day 1 after littering) x 100;
Lactation index (%) = (Number of live offspring on Day 13 after littering/ Number of live offspring on Day 4 (after blood sampling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item-related signs associated with dose administration were limited to transient incidences of paddling of the forepaws in all groups of treated males and females.
Throughout the treatment period, all groups of treated animals showed chin rubbing on return to the home cage, and animals given 1000 mg/kg bw/day showed salivation; females given 330 mg/kg bw/day also showed salivation during the lactation period. These signs are considered to reflect distaste of the treated formulations and are not indicative of a toxic response.
There was no test item-related change in general clinical condition observed throughout the study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test article-related death was noted.
A female receiving 330 mg/kg/ bw/day, was euthanized during gestation for welfare reasons, due to dosing difficulties, on Day 19 of treatment. Another female at 330 mg/kg bw/day and a high dose group female were euthanized on Day 25 and Day 26 respectively, because they failed to litter and were subsequently found not to be pregnant. There was no macroscopic or microscopic observation that could explain the apparent infertility in these females. All macroscopic and microscopic findings were considered spontaneous and/or incidental.

These unscheduled deaths of females were not considered to be related to the administration of Y-15853.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower than control mean body weight gain was evident in F0 males receiving 1000 mg/kg bw/day throughout the treatment period, such that overall group mean body weight gain during Weeks 0-4 was 42% lower than control. The body weight performance of all other F0 male groups was considered unaffected by treatment.
The body weight gain before mating and during gestation of F0 females of all groups was
unaffected by treatment.
Higher than control mean body weight gain was evident in the F0 females receiving 1000 mg/kg bw/day during Days 7-13 of lactation, with overall mean body weight gain during Days 1-13 of lactation being 79% higher than control. Mean body weight gain during lactation for females given 100 or 330 mg/kg bw/day was unaffected.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was slightly lower than control during the 2-week pre-pairing treatment period in all F0 male groups, most notably during Week 1 in males receiving 1000 mg/kg bw/day where mean food intake was 22% lower than control, attaining statistical significance.
Food consumption was generally considered unaffected in all F0 females when compared with controls before mating, during gestation and lactation, with the exception of slightly lower than control food consumption in females receiving 1000 mg/kg bw/day during the first week of the pre-pairing period (14% lower than control) and statistically significantly lower than control during Days 14-20 of gestation (11% lower than control).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The mean serum T4 concentrations of F0 adult male animals in the low and the high dose group were slightly lower than controls, however, no effect of treatment was inferred. Detailed results are attached below.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There was no treatment-related microscopic finding.
At terminal necropsy, extensive chronic inflammation of the thoracic cavity was seen in one male receiving 1000 mg/kg bw/day. This finding correlated with macroscopic thoracic cavity adhesions involving multiple organs and abnormal red fluid. This finding was considered likely to be related to the gavage procedures.
All microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Han Wistar rats of this age; therefore, they were considered not test article-related.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle regularity before and during treatment, pre-coital interval, mating performance,
fertility and gestation length, and the number of copulation plugs and sperm count estimates
at mating were considered unaffected by treatment.
All F0 females were in diestrus of the day of termination; this is as expected since the females were still nursing the offspring.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive performance:
no effects observed
Description (incidence and severity):
Percentage mated was 100% for all groups. The conception rate was 100% for the controls and the low dose groups, and 90% for the mid and the high dose group. The fertility rate was 100%, 100%, 90% and 90% for the controls and the low, mid and high dose groups, respectively.

The gestation index was 100%, 100%, 89% and 100% for the controls and the low, mid and high dose groups, respectively.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: -

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed that were considered to be caused by exposure to the test item.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Litter size, sex ratio and survival indices were considered unaffected by treatment.
There were differences, some that achieved statistical significance, such as slightly higher than control litter size and offspring sex ratio in females receiving 1000 mg/kg bw/day, however, due to the small magnitude of change and nature of the differences they were considered incidental.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean birth weight of the offspring was similar in all groups and unaffected by parental treatment with Y-15853.
At 1000 mg/kg bw/day, lower than control mean body weight gain was evident throughout Days 1-13 of lactation in male and female F1 offspring , most notably during Days 7-13 of age, coinciding with the period of increased maternal body weight gain in this group. Overall mean offspring body weight gain was subsequently 30% lower than control in both sexes.
Offspring body weight gain at 100 or 330 mg/kg bw/day was unaffected.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The mean serum T4 concentrations of F1 male and female offspring on Day 13 of age were essentially similar to concentrations measured in control offspring.
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
On Day 1 of age, F1 male offspring derived from F0 females given 1000 mg/kg bw/day and F1 female offspring in all treated groups showed a slight, but statistically significant increase in ano-genital distance when compared to controls. There was no dose response relationship observed in the F1 female offspring.

In the absence of a dose response relationship and noting that the differences from control were small, and taking into account that there was no other finding indicative of endocrine disruption, it was concluded unlikely that the slight increase in ano-genital distance was of toxicological significance.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Male offspring did not develop nipples.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no treatment-related macroscopic finding.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
330 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: -

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
A screening study for reproduction and developmental effects was performed according to OECD guideline 421 and following GLP principles. Based on the results it is concluded that the parental NOAEL is 1000 mg/kg bw/day, and the NOAEL for development is 330 mg/kg bw/day.
Executive summary:

A screening test for reproductive/developmental effects was performed according to OECD/EC guidelines and GLP principles. Three groups of ten male and ten female Han Wistar rats received Y-15853 at doses of 100, 330 or 1000 mg/kg bw/day by oral gavage administration. Males were treated daily for two weeks before pairing and throughout pairing up to necropsy for a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, dried and de-acidified corn oil, at the same volume dose as treated groups.


During the study, clinical condition, body weight, food consumption, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.


Analysis of formulations showed animals were administered an accurate and homogenous dose. There was no difference in T4 levels that was considered to be treatment-related.


There was no test item-related change in general clinical condition observed throughout the study. Lower than control mean body weight gain was evident in F0 males receiving 1000 mg/kg bw/day throughout the treatment period. The body weight gain before mating and during gestation of F0 females of all groups was unaffected by treatment, however, higher than control mean body weight gain was evident in the F0 females receiving 1000 mg/kg bw/day during Days 7-13 of lactation. Food consumption was slightly lower than control during the 2-week pre-pairing treatment period in all F0 male groups. Food consumption was unaffected in all F0 females before mating, during gestation and lactation, with the exception of slightly lower than control food consumption in females receiving 1000 mg/kg bw/day during the first week of the pre-pairing period and during Days 14-20 of gestation.


Estrous cycle regularity before and during treatment, pre-coital interval, mating performance, fertility and gestation length, and the number of copulation plugs and sperm count estimates at mating were considered unaffected by treatment.


Organ weights were considered unaffected by treatment and there was no treatment-related macroscopic finding. There was no treatment-related microscopic finding.


Litter size, offspring survival, sex ratio, birth weights, general condition and macropathology showed no adverse effect of parental treatment. Ano-genital distance on Day 1 of age was slightly increased in male offspring at 1000 mg/kg bw/day and in all groups of female offspring. Body weight gain was markedly lower than control throughout lactation for male and female F1 offspring at 1000 mg/kg bw/day.


Overall the results indicate that there was no effect on reproductive performance. Lower than control body weight gain in the F0 males given 1000 mg/kg bw/day, associated with lower than control food consumption during the pre-pairing period, had no impact on the survival, general clinical condition, reproductive performance, organ weights or macro/micropathology and in isolation, within the context and scope of this study, were considered non-adverse. F1 offspring growth was adversely affected by parental treatment at 1000 mg/kg bw/day, resulting in a 30% reduction in weight gain during Days 1-13 of age.


Based on the results of this study, it is concluded that the NOAEL for reproductive/developmental toxicity was 1000 mg/kg bw/day for the parental animals and 330 mg/kg bw/day for the F1 offspring.