2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Toxicity to reproduction, OECD guideline 421 (Charles River, 2021, GLP, K1)

Administration by oral gavage to male and female rats (10/sex/dose) at dose-levels of 25, 50 and 75 mg/kg bw/day.

NOAEL (systemic toxicity) = NOAEL (mating and fertility) = NOAEL (developmental toxicity)= 75 mg/kg bw/day, no treatment related-effects observed up to 75 mg/kg bw/day.

 

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 August 2020 To 23 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 421.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Version 29 July 2016

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions. See appendix 1 of the study report available in the attached document.

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Storage Conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C, protected from light, under nitrogen.

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The Crl:WI(Han) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC.
- Age at study initiation: Approximately 11 - 12 weeks old
- Weight at study initiation: 169 to 215 g for females and 216 to 302 g for males.
- Housing: On arrival, animals were group housed (2 - 3 animals of the same sex) until cohabitation. During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in clean, solid-bottom cages with bedding material (Bed O’Cobs® or other suitable material).
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal, ad libitum.
- Water (e.g. ad libitum): Municipal tap water, ad libitum.
- Acclimation period: minimum of 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 68°F to 78°F (20°C to 26°C)
- Humidity: 30% to 70%
- Air changes: no data
- Photoperiod: 12 hrs dark / 12 hrs light

- Environmental Enrichment
Enrichment devices/supplements will be provided to each animal for environmental enrichment and to aid in maintaining the animals’ oral health, beginning during acclimation and continuing throughout the course of the study.

IN-LIFE DATES: From 11 August to 08 November, 2020

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: test substance dosing formulations stored refrigerated (target of 5°C), protected from light, and purged with nitrogen, until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): used in previous studies.
- Amount of vehicle (if gavage): 2.5 mL/kg
- Lot/batch no. (if required): 2HK0034
- Purity: 100%

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: For a maximum of fourteen days.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually during gestation and lactation, in clean, solid-bottom cages with bedding material (Bed O’Cobs® or other suitable material).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance formulations have been previously shown to be stable and homogeneous over the range of concentrations used on this study for at least 8 days refrigerated (2-8℃) (Charles River, Study No. 008100281).

Duration of treatment / exposure:

- Males: daily for 14 days prior to mating and continuing through 1 day prior to euthanasia (Study Days 0–27).
- Females: daily for 14 days prior to mating and continuing through Lactation Day 12.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control (Group 1)

Dose / conc.:

25 mg/kg bw/day

Remarks:

Low dose (Group 2)

Dose / conc.:

50 mg/kg bw/day

Remarks:

Mid dose (Group 3)

Dose / conc.:

75 mg/kg bw/day

Remarks:

High dose (Group 4)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage selection for this study was based on two previous 28-day repeated dose studies in Wistar Han rats (C32123, C00641)2,3 in which dose levels of 5, 25, and 50 mg/kg/day and 100, 300, 600, and 1000 mg/kg/day were tested. Based on the results of the studies, a NOAEL of 50 mg/kg/day was established. Dose levels of >= 100 mg/kg/day were considered to have exceeded the no-adverse-effect-level due to test substance related and adverse effects on clinical biochemistry indicative of impaired kidney function and anatomical pathological findings of myocardial necrosis and hepatocellular vacualation in the liver noted at this dose level. Based on these results 0, 25, 50 and 75 mg/kg/day dose levels were selected. The high-dosage level is expected to produce some toxicity, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal toxic effects. The low-dosage level is expected to produce no observable indications of toxicity.
- Rationale for animal assignment (if not random): random.

Positive control:

Not applicable.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily throughout the study. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded approximately 2 hours postdose.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 3, 7, 10, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 10, and 13.

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. Food consumption was quantitatively measured weekly until cohabitation. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 3, 7, 10, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, and 13.

OTHER EXAMINATIONS
Thyroid Hormone Analysis: samples were analysed for Thyroxine (Total T4) in males and females for all groups at Study Day 28 and lactation Day 13.

Oestrous cyclicity (parental animals):

For all females, daily vaginal lavages will be performed to determine the stage of estrus beginning during the pretest period and continuing through the 2-week premating
and mating treatment periods until evidence of mating is observed. Lavaging will continue for those females with no evidence of mating until termination of the mating period. A vaginal lavage will also be performed on the day of necropsy.

Sperm parameters (parental animals):

Parameters examined in P male: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

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, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: yes

OTHER EXAMINATION:
Thyroid Hormone Analysis: Blood samples were analyzed for Thyroid Hormone Thyroxine (Total T4):
- At least 2/Litter, PND 4 (all groups)
- 2/sex/Litter, PND 13 (all groups).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 6 weeks of treatment
- Maternal animals: All surviving animals on Day 13 of lactation

GROSS PATHOLOGY: Yes
- Necropsy: Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss.
The organs identified in Table 7.8.1/1 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

HISTOPATHOLOGY: Yes.
Tissues identified in Table ( Table 7.8.1/2) for microscopic examination were evaluated from all animals in the control and high-dose groups.

Postmortem examinations (offspring):

SACRIFICE
- Selected F1 offspring were sampled and killed on Day Day 13, surviving animals were euthanized via an intraperitoneal injection of sodium pentobarbital.

GROSS NECROPSY
- On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. The organs (Thyroid (with parathyroids, if present) were weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia.

Statistics:

- Parametric/Non-parametric: Levene’s test will be used to assess the homogeneity of group variances. The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis
test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s or
Dunn’s test, respectively.
- Non-Parametric:
The groups will be compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis
test is found to be significant, then the above pairwise comparisons will be conducted using
Dunn’s test.
- Incidence: A Fisher’s exact test will be used to conduct pairwise group comparisons of interest.
Parametric
- The groups will be compared using an overall one-way ANOVA F-test. If the overall F-test is
found to be significant, then pairwise comparisons will be conducted using Dunnett’s test.

Reproductive indices:

Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or no confirmed mating date and pregnant)/No. of Males (Females) Paired x 100.

Male Fertility Index (%)= No. of Males Impregnating a Female/No. of Males with Evidence of Mating (or Females Confirmed Pregnant) x 100

Male Pregnancy Index (%) = Number of Males Impregnating a Female/Number of Paired Males x 100

Female Fertility Index (%) = Number of Pregnant Females/Number of Females with Evidence of Mating (or no x confirmed mating date and pregnant) x 100

Female Pregnancy (Index %) = Number of Females Pregnant/Number of Females that were paired x 100.

Offspring viability indices:

Live birth index (%) = Number of live offspring on Day 1 after littering x 100 / Total number of offspring born

Viability index (%) = Number of live offspring on Day 4 x 100 / Number live offspring on Day 1 after littering

Lactation index (%) = Number of live offspring on Day 13 after littering x 100 / Number of live offspring on Day 4 postpartum

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related clinical observations were noted during the generation at the daily examinations or at 2 hours postdosing. Clinical observations noted in the test substance-treated groups were transient, occurred infrequently, and/or in a manner that was not dose-response.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related effects on survival at any dose level. One female in 25 mg/kg/day group had a total litter loss and was subsequently euthanized on Lactation Day 1. One female in the control and one female in the 25 mg/kg/day group were euthanized on Postmating or Postcohabitation Day 25.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

MALES
Mean body weights and body weight gains in the 25, 50, and 75 mg/kg/day group males were unaffected by test substance administration throughout the study. None of the differences from the control group were statistically significant.
FEMALES
- Weekly: Mean body weights and body weight gains in the 25, 50, and 75 mg/kg/day group females were unaffected by test substance administration during the premating period. None of the differences from the control group were statistically significant.
- Gestation: Mean body weights and body weight gains in the 25, 50, and 75 mg/kg/day groups were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.
- Lactation: Mean body weights and body weight gains in the 25, 50, and 75 mg/kg/day groups were unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

MALES
Mean food consumption in the 25, 50, and 75 mg/kg/day group males was unaffected by test substance administration throughout the premating dosing period. None of the differences from the control group were statistically significant.
FEMALES
- Weekly: Mean food consumption in the 25, 50, and 75 mg/kg/day group females was unaffected by test substance administration during the premating period. None of the differences from the control group were statistically significant.
- Gestation: Mean food consumption in the 25, 50, and 75 mg/kg/day groups was unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.
- Lactation: Mean maternal food consumption in the 25, 50, and 75 mg/kg/day groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion efficiency was considered to have been unaffected by treatment for both sexes throughout the study, which included for females, gestation and lactation phases, at 25, 50 and 75 mg/kg bw/day.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F0 males in the 25, 50, and 75 mg/kg/day groups. Differences from the control group were slight and not statistically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment-related effects observed at all tested doses.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at any dose level. No statistically significant differences were noted between the control and test substance-treated groups. 1, 1, 0, and 0 mating pairs in the control, 25, 50, and 75 mg/kg/day groups, respectively, did not produce a litter.
The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean number and length of estrous cycles in these groups were also similar to the control group values. None of these differences were statistically significant.

Analyses of Dosing Formulations:
The analyzed dosing formulations contained 98.8% to 108% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

REPRODUCTIVE PERFORMANCE: FERTILITY
There was no adverse effect of treatment on fertility at all tested doses.

GESTATION LENGTH
Mean gestation lengths and gestation index in the 25, 50, and 75 mg/kg/day groups were similar to the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups. The mean postimplantation loss (unaccounted-for sites) and implantation sites in the 25, 50, and 75 mg/kg/day groups were similar to the control group values.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance-related effects were noted for estrous cyclicity, reproductive performance (mating, fertility, and pregnancy indices and precoital interval) or delivery observations (including gestation length, gestation index, and implantation sites)

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on survival, clinical condition, body weight gain, food consumption or macroscopic necropsy findings and subsequent microscopic evaluation

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance administration.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One (1), 3(3), 2(2), and 2(1) pups (litters) in the control, 25, 50, and 75 mg/kg/day groups, respectively, were found dead or euthanized in extremis.

Litter Data and Postnatal Survival:
The total number of newborn pups, number of live newborn pups, and the percentage of males at birth in the 25, 50, and 75 mg/kg/day groups were similar to the control group values. Postnatal survival, including live birth index (survival from PND 0), viability index (survival from PND 0–4), and survival index (survival from PND 4–13), in the 25, 50, and 75 mg/kg/day groups were unaffected by test substance administration.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean male and female birth weights in the 75 mg/kg/day group were generally comparable to the control group on PND 1. During PND 1–13, slightly lower mean male and female pup body weight gains were observed for the 75 mg/kg/day group; differences were not statistically significant versus the control group. As a result, slightly lower (not statistically significant) mean pup body weights were observed in this group from PND 4 through PND 13. Due to the deficits
in mean body weight gains, mean absolute body weights at termination (PND 13) were 6.30% and 7.07% lower than the controls for males and females, respectively; differences were not statistically significant. However, based on the low magnitude of difference from the control group, the pup body weight effects were considered test substance-related but nonadverse.
Mean male and female pup body weights and body weight gains in the 25 and 50 mg/kg/day groups were unaffected by parental test substance administration throughout the postnatal period (PND 1–13).

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

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 25, 50, and 75 mg/kg/day groups on PND 1 and 4 were similar to the control group values. Differences from the control group were slight and not statistically significant, with the following exception. Statistically significantly higher mean anogenital distances (absolute and normalized) were noted for females in the 75 mg/kg/day group compared to the control group on PND 1. These differences were not considered test substance-related because the values for females in this group were comparable to the control group on PND 4, and therefore the differences observed on PND 1 were attributed to natural variation.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen retention in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. There were no retained nipples in any group.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pup thyroid weights were unaffected by test substance-administration. Differences from the control group were not statistically significant. Lower mean thyroid/parathyroid weights were noted for female pups in the 75 mg/kg/day group, but similar effects were not observed in male pups on PND 13 and there were no correlating effects on thyroid hormone values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One (1), 3(3), 2(2), and 2(1) pups (litters) in the control, 25, 50, and 75 mg/kg/day groups, respectively, were found dead or euthanized in extremis. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead or euthanized in extremis. In the 50 mg/kg/day group, Pup No. 3509-01 was noted with a right-sided aortic arch, left origin subclavian artery, misshapen snout, absent eyes, cleft
lips, absent left ovary, oviduct and uterine horn, exencephaly, and an absent left kidney. These malformations were not considered test substance-related as they were noted in a single fetus in a manner that was not dose-responsive.

At necrospy: No internal findings that could be attributed to parental test substance administration were noteat the necropsy of pups euthanized on PND 13. A single pup in the 75 mg/kg/day group was noted with an enlarged and pale thyroid gland. Because this finding was noted in a single high-dose pup and was not noted in the female pups at this dose level, the finding was not considered test substance-related. There were no other noted findings at necropsy.

Organ Weights: Mean pup thyroid weights were unaffected by test substance-administration. Differences from the control group were not statistically significant. Lower mean thyroid/parathyroid weights were noted for female pups in the 75 mg/kg/day group, but similar effects were not observed in male pups on PND 13 and there were no correlating effects on thyroid hormone values.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis (PND 13):
There were no test substance-related effects on thyroid hormone (T4) values in the F1 males and females at any dose level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were detected

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the test conditions of this study, in the absence of any effect on reproductive parameters or any evidence of adverse toxicity for F0 males and females, a dose level of 75 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity and F0 systemic toxicity when the test substance was administered orally by gavage to rats. In addition, in the absence of any adverse effects on any F1 parameters, the NOAEL for F1 neonatal toxicity was considered to be 75 mg/kg/day.

Executive summary:

In this Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 421 and in compliance with GLP, the test item was administered once daily by oral gavage to three groups, each of ten male and ten female Crl:WI(Han) strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 25, 50 and 75 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (100% polyethylene glycol (PEG) 300).

Animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing through 1 day prior to euthanasia (Study Days 0–27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 12.

 

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, thyroid hormones, macroscopic findings, organ weights, and histopathologic examinations.

 

There were no test substance-related effects on survival for F0 males and females at any dose level. There were no test substance-related clinical observations at the daily examinations or approximately 2 hours following dose administration for males and females at any dose level.

For F0 males and females, mean body weights, body weight gains, and food consumption throughout the treatment period, including during gestation and lactation for females, were unaffected by test substance administration at 25, 50, and 75 mg/kg/day.

No test substance-related effects were noted for estrous cyclicity, reproductive performance (mating, fertility, and pregnancy indices and precoital interval) or delivery observations (including gestation length, gestation index, and implantation sites) at any dose level.

There were no test substance-related effects on T4 concentrations for F0 males at 25, 50, and 75 mg/kg/day.

There were no test substance-related effects on organ weights, gross necropsy findings, and histopathologic findings at any dose level.

 

The mean numbers of F1 pups born (including number of live newborn pups), percentage of males at birth, postnatal survival (including live birth, viability, and survival indices), clinical condition of the pups, anogenital distance, and areola/nipple retention (males) in the 25, 50, and 75 mg/kg/day groups were unaffected by test substance administration. In the 75 mg/kg/day group, higher mean anogenital distances were noted for females relative to the control group on PND 1. However, mean anogenital distances on PND 4 were comparable to the control group and as such, the differences on PND 1 were attributed to natural variation.

In the 75 mg/kg/day group F1 males and females, slightly lower mean pup body weight gains were noted during PND 1–13, resulting in lower (6.30% and 7.07%, respectively) mean pup body weights on PND 13. These changes were considered test substance-related but non adverse, due to the low magnitude of change from the control group. There were no test substance-related effects on mean pup body weights and body weight gains at 25 and 50 mg/kg/day.

There were no test substance-related macroscopic findings noted in F1 pups that were found dead or at the scheduled necropsy on PND 13. There were no test substance-related effects on serum T4 levels or thyroid/parathyroid weights in the F1 pups at any dose level on PND 13.

 

Under the conditions of this screening study, in the absence of any effect on reproductive parameters or any evidence of adverse toxicity for F0 males and females, a dose level of 75 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity and F0 systemic toxicity when the test item was administered orally by gavage to Crl:WI(Han) rats. In addition, in the absence of any adverse effects on any F1 parameters, the NOAEL for F1 neonatal toxicity was considered to be 75 mg/kg/day.

Based on this study, the test material is not classified for developmental or reproductive toxicity according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is acceptable and satisfies the requirement for a reproduction/developmental toxicity screening test in rats.

 

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Only one study available, GLP-compliant and of high quality (Klimisch score = 1)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A key study was identified (Charles River, 2021, Rel.1).

In this reproduction/developmental toxicity screening test conducted in accordance with OECD test guideline No 421 and in compliance with GLP, rats were administered the test material at dose levels of 0, 25, 50 and 75 mg/kg bw/day for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).

 

There were no unscheduled deaths. No clinical signs of toxicity, no adverse effects on body weight change, and no adverse effects on dietary intake were detected.Daily visual inspection of water bottles did not reveal any overt intergroup differences in water intake.

No treatment-related effects were detected in mating performance, on fertility and in the length of gestation between control and treated groups.

No differences in litter size, sex ratio or viability parameters were detected for litters from treated animals when compared to those from controls.

No significant differences in litter weights were evident in the treated groups when compared to controls. There were no clinically observable signs of toxicity observed in offspring.

No treatment-related macroscopic abnormalities were detected for adults or offspring.

No treatment-related changes were detected for testes and epididymis weights for treated males when compared to controls.

No treatment-related histopathology effects were detected.

 

Under the conditions of this screening study, in the absence of any effect on reproductive parameters or any evidence of adverse toxicity for F0 males and females, a dose level of 75 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity and F0 systemic toxicity when the test substance was administered orally by gavage to Crl:WI(Han) rats. In addition, in the absence of any adverse effects on any F1 parameters, the NOAEL for F1 neonatal toxicity was considered to be 75 mg/kg/day.

Effects on developmental toxicity

Description of key information

Since no alerts for developmental toxicity were observed in the screening study, no additional study is proposed at this tonnage level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Only one study available, GLP-compliant and of high quality (Klimisch score = 1)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no developmental toxicity / teratogenicity studies on the test material. The reproductive/development toxicity screening test described above showed no-observed-effect-level for offspring developmental toxicity at 75 mg/kg bw/day, the highest concentration level tested.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data on the source substance, no additional classification is proposed for the target substance according to the Regulation (EC) No 1272/2008 and to the GHS based on the absence of adverse effects observed during the OECD 421 screening study.