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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:
2018 - 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Batch No.: 2310016

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (spermidentification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed.
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy, for a total of 28/29 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum for a total of 50 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Frequency of treatment:
Once a day for 14 days (males).
Once a day for 50 days (females).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
Each group comprised 10 male and 10 female rats.
Control animals:
yes

Examinations

Oestrous cyclicity (parental animals):
Oestrous cycle did not show relevant differences between control and treated groups.
Mating was not detected in one female receiving 30 mg/kg/day (no. X0120025 mated with male no. X1020026). This female gave birth and was subsequently sacrificed at termination on Day 14 post partum.
Pre-coital intervals, copulatory index and fertility index did not show differences that could be considered related to treatment.
Litter observations:
No significant differences in total litter size, live litter size, pup loss, mean pup weight and litter weights were observed among treated and control females at birth and on Days 1, 4 and 13 post partum.
Sex ratios at birth, on Days 4 and 14 post partum did not show differences between groups, when calculated as the percentage of males.
Postmortem examinations (parental animals):
No changes were observed on terminal body weight of treated animals, when compared to the controls. No relevant changes were reported in the absolute and relative organ weights of treatment groups of both sexes, when compared to control data, with the exception of the statistically significant increase of relative ovaries weight of the high dose female group.
However, since the histopathological evaluation of ovaries was comparable to control animals, no toxicological significance could be attributed to this finding.

Macroscopic observations
Animals that completed the treatment period and killed at termination did not showrelevant macroscopic changes that could be considered treatment-related.
The sporadic changes, such as enlarged ovaries (no. X1020069) or hairloss of forelimbs, hindlimbs and ventral region (no. X1020065) in two high dose treated females were considered spontaneous and incidental, also observed in untreated animals under our experimental conditions and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Microscopic observations
No treatment-related changes were noted. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
The sporadic lesions were considered to be an expression of spontaneous and/or incidental pathology seen in this species.
Postmortem examinations (offspring):
Necropsy findings in decedent pups, in pups sacrificed on Days 4 and 14 post partum and nipple count

All organs autolysed or no abnormalitieswere observed in decedent pups.
No abnormalities were recorded in pups sacrificed on Days 4 or 14 post partum with the exception of tip of tail missing in one pup of female no. X1020027 (Group 2) and moderate hairloss of muzzle noted in pups of female no. X1020041 (Group 3).
No nipples were observed in male pups on Day 14 post partum.

Pups thyroid weight on Day 14 post partum
No significant differences were noted in thyroid weight between control and treated pups.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No relevant clinical signs were detected at the daily observation of the animals.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Description (incidence):
No mortality occurred throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences in bodyweight and bodyweight gainwere noted between control and treated males and females throughout the study.
The statistically significant increase in body weight gain of high dose males and low dose females respect to the control during the pre-pairing period was not considered of toxicological relevance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption were observed in both sexes.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Thyroid hormones

Parental males
No relevant changes were recorded.
Mean thyroid stimulating hormone value was higher than controls in males dosed at 500 mg/kg/day. However, this finding was considered to be unrelated to treatment.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
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):
Oestrous cycle did not show relevant differences between control and treated groups.
Mating was not detected in one female receiving 30 mg/kg/day (no. X0120025 mated with male no. X1020026). This female gave birth and was subsequently sacrificed at termination on Day 14 post partum.
Pre-coital intervals, copulatory index and fertility index did not show differences that could be considered related to treatment.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
reproductive function (oestrous cycle)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No compound-related effects were observed. Pre-weaning clinical signs were comparable between treated and control groups or considered incidental.
In addition, found dead and/or missing pups were also observed both in control and treated groups, with similar incidence.
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
Found dead and/or missing pupswere also observed both in control and treated groups, with similar incidence.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Pups thyroid weight on Day 14 post partum
No significant differences were noted in thyroid weight between control and treated pups.
Urinalysis findings:
no effects observed
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance
No relevant changes were seen between the control and treated pups in anogenital distance.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were observed in male pups on Day 14 post partum.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
All organs autolysed or no abnormalitieswere observed in decedent pups. No abnormalities were recorded in pups sacrificed on Days 4 or 14 post partum with the exception of tip of tail missing in one pup of female no. X1020027 (Group 2) and moderate hairloss of muzzle noted in pups of female no. X1020041 (Group 3).
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 500 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
clinical signs
mortality
body weight and weight gain
clinical biochemistry

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
500 mg/kg bw/day

Applicant's summary and conclusion

Conclusions:
In conclusion, no signs of treatment-related toxicity were observed following treatment with the test substance, when administered to rats by oral route at dose levels of 30, 100 and 500 mg/kg/day, at any of the dose levels investigated. Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 500 mg/kg/day for males and females.
Executive summary:

The toxic effects in Sprague Dawley rats of both sexes after repeated dosing with the test substance, as well as any effects of the test item on male and female reproductive performance (such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation) were investigated in this study. All doses (30, 100, 500 mg/kg/day) were administered orally by gavage. The control group received softened water. Males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 28/29 days. Females were treated for 14 days prior to

pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for at least 50 days.

No mortality occurred and all females had live pups on Day 14 post partum with the exception of one not pregnant female in Group 4.

No clinical signs were observed during the study in parental males and females.

No differences in body weight and food consumption were observed in treated animals, compared to the control group.

Thyroid hormones investigation performed in parental males and in pups did not reveal changes that could be considered treatment-related.

No treatment related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment related differences among treated and control groups.

Parturition, lactation, implantation, litter data and sex ratio did not show any changes of toxicological relevance.

No significant differences in the anogenital distance (normalised value) were seen between control and treated groups both for male and female pups.

No nipples were observed in male pups.

Necropsy findings and thyroidweight in pups did not reveal any treatment-related effect. In parental animals, no treatment-related changes were observed at post mortem in terminal body weight, organ weights, macroscopic and microscopic observations.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages and a regular layering in the germinal epithelium was described.

In conclusion, no signs of treatment-related toxicity were observed following treatment with the test substance, when administered to rats by oral route at dose levels of 30, 100 and 500 mg/kg/day, at any of the dose levels investigated. Based on the results of the present study, theNOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 500 mg/kg/day for males and females.