p-tert-butylstyrene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In-life phase: 07 June 2017 to 01 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/Batch no.: 122016TBSSV
Purity: 95.9%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age at first dose: 11-12 weeks
Weight at first dose: Males-390-465 g and Females-239.1-292.6 g

Animals were acclimated to laboratory conditions for at least five days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum.
Water: Filtered water was provided ad libitum via an automatic watering system supplemented with water bottles as needed.
Housing: Animals were housed in one room in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information.
Temperature: 20 to 26°C
Humidity: 30-70%
Light-dark cycle: 12-hour light/12-hour dark
Air changes: Minimum of 10 air changes per hour

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

The vehicle/control substance was considered 100% pure for formulation purposes. The test substance was used as received and formulated with no adjustments. The vehicle/control substance, peanut oil, was used as received; no formulations were necessary. Formulations for Groups 2 (5 mg/mL/10 mg/kg bw/day), 3, (15 mg/mL/30 mg/kg bw/day) 4 (30 mg/mL/60 mg/kg bw/day) and 5 (50 mg/mL/100 mg/kg bw/day) were prepared weekly by adding the appropriate amount of the control test substance to the required amount of vehicle/control substance and mixinged until visually uniform. Formulations were stored in a refrigerator (2-8 degree Celsius) until used for dosing.

Details on mating procedure:

Animals were individually housed prior to cohabitation and after confirmation of mating. During cohabitation, one male and one female from the same group were housed together. After at least 14 days of vaginal lavage and dosing, one female from each group was cohabited with one male from the corresponding group (1:1). Females assigned to 60 mg/kg bw/day were cohabitated with males assigned to 100 mg/kg bw/day group. The females were tested daily by vaginal lavage beginning on the day following cohabitation. The animals were separated when mating was confirmed, or after 14 days, and observed as indicated above. Day 0 of pregnancy (gestational day (GD) 0) was defined as the day on which mating evidence is was confirmed (vaginal plug or presence of sperm). The stage of estrus was determined in each female for at least 14 days prior to randomization, 14 days prior to cohabitation and during cohabitation until evidence of mating, or 14 days of cohabitation, whichever occurred first.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analysis of the dose formulations was performed as part of the method validation. Samples were analyzed for test substance concentration using a validated method. Since all dose formulations were solutions; therefore, homogeneity analysis was not required.

Duration of treatment / exposure:

The animals were dosed via oral gavage at a volume of 2 mL/kg bw. Dosing volumes were based on the most recent body weights. F0 males were dosed for at least 28 days. The F0 females were dosed for two weeks prior to cohabitation, during cohabitation, through gestation, and to at least Postnatal Day (PND) 12. The first day of dosing was designated as Day 1 for each animal.

Frequency of treatment:

Once daily

Details on study schedule:

F0 males were dosed for at least 28 days. The F0 Females were dosed for two weeks prior to cohabitation, during cohabitation, through gestation, and to at least postnatal day (PND) 12. The first day of dosing was designated as SD1 for each animal.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

for females only

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

for males only

Parental animals: Observations and examinations:

Post dosing, F0 males and females were examined regularly for mortality, moribundity, general health, body weight, food consumption and signs of toxicity. Physical examinations included evaluations of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behavior patterns. Prior to scheduled necropsy, routine hematological, clinical chemistry and hormonal analysis were done.

Oestrous cyclicity (parental animals):

The stage of estrus was determined in each female for at least 14 days prior to randomization, 14 days prior to cohabitation and during cohabitation until evidence of mating, or 14 days of cohabitation, whichever occurred first.

Litter observations:

Following delivery, the F1 generation pups were observed for litter size, sex, weight, anogenital distance and nipple retention (in male pups) and clinical signs. On PND 4 and 13 9before scheduled necropsy), blood samples were collected, pooled and analysed for hormone levels.

Postmortem examinations (parental animals):

Gross necropsy of F0 animals included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. For all F0 females, the number of implantation sites was recorded. All reproductive organs (testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix; sex appropriate) and thyroids from all F0 animals were collected and weighed as soon as possible after dissection; paired organs were weighed together.

Histopathological examination of tissues from the selected animals in the control and high dose group revealed test substance-related changes in the bone marrow (femur) of females only, the pancreas, thyroid and thymus in males only, and the kidney and liver in both sexes. Therefore, the following tissues from selected animals in 10 mg/kg bw/day and 30 mg/kg bw/day were processed to slides and examined microscopically: bone marrow (femur, females only, 5/group); pancreas, thyroid, and thymus (males only, 5/group); kidney and liver (both sexes, 5/sex/group).

Postmortem examinations (offspring):

All pups, except those assigned to blood collection, were examined externally for gross abnormalities.

Statistics:

See under "any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

seminal vesicle

testes

Treatment related:

yes

Dose response relationship:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

Organ:

kidney

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

other: general systemic toxicity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Conclusions:

Under the study conditions, the LOAEL for systemic toxicity in parental male ratss was determined to be 10 mg/kg bw/day (due to microscopic findings in thyroid). The NOAEL for systemic toxicity in female rats was considered to be 10 mg/kg bw/day due to adverse body weight changes, clinical chemistry, organ weight and microscopic findings. The NOAEL for reproductive toxicity in both male and female rats was determined to be 10 mg/kg bw/day dose. The NOAEL for fetal developmental toxicity was determined to be 10 mg/kg bw/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats according to OECD Guideline 422, in compiance with GLP. Male rats were administered the test substance at doses of 0, 10, 30 or 100 mg/kg bw/day for 14 d prior to cohabitation and during cohabitation for at least a total of 28 d. Female rats were dosed with the test substance at doses of 0, 10, 30 or 60 mg/kg bw/day prior to cohabitation, during cohabitation, through pregnancy, and throughout lactation to Postnatal Day 13 (PND 13). Treatment-related effects in male rats included a significant effect on the body weight, body weight changes, organ weights, and microscopic findings. For female rats, repeat dose toxicity was observed at doses of 30 and 60 mg/kg bw/day primarily due to reduced body weight and microscopic findings in liver, kidney and thyroid. Important microscopic findings consisted of cytoplasmic alteration in hepatocytes at doses greater than 30 mg/kg bw/day in both sexes, thyroid follicular cell hypertrophy in males from 10 mg/kg bw/day and vacuolation of distal tubules in the kidney in males and females at the respective high doses. The test substance induced reproductive toxicity at doses of 30, 60 (females only) and 100 (males only) mg/kg bw/day. Treatment-related degeneration/atrophy of seminiferous tubules in all high dose males resulted in complete loss of fertility at this dose. Although degeneration of spermatids was only identified in one animal administered 30 mg/kg bw/day, this change was associated with decreased fertility and fecundity; therefore, testicular changes were adverse at or above 30 mg/kg bw/day. Decreases in testes and epididymal weight correlated with these changes in the high dose males. In females at 60 mg/kg bw/day, no viable pregnancy was observed and pups could not be evaluated. This was likely due to the effect on spermatogenesis, as there was evidence of copulation and normal estrus cyclicity at the time of cohabitation. The copulation index, fertility index and pregnancy rate are significantly and dose-dependently reduced due to the treatment-related effects in males. The test substance also induced developmental toxicity at 30 mg/kg bw/day evidenced by growth inhibition and reduced litter size without any indication of induced specific malformations. Therefore, the reduced pup growth and litter size could potentially be due to maternal and/or paternal toxicity rather than direct impairment of development in utero, but the potential for association between the treatment and direct impairment of in utero development cannot be ruled out based upon the results of this study. The dose group of 60 mg/kg bw/day could not be evaluated for developmental toxicity due to dams not producing any litters. Under the study conditions, the LOAEL for systemic toxicity in parental male rats was determined to be 10 mg/kg bw/day (due to microscopic findings in thyroid). The NOAEL for systemic toxicity in female rats was considered to be 10 mg/kg bw/day due to adverse body weight changes, clinical chemistry, organ weight and microscopic findings. The NOAEL for reproductive toxicity in both male and female rats was determined to be 10 mg/kg bw/day dose. The NOAEL for fetal developmental toxicity was determined to be 10 mg/kg bw/day (Murphy, 2019b).

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

described adverse effects in available studies occure in presence of maternal toxicity as well

and are rated rather unspecific.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In-life phase: 07 April 2018 to 27 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/Batch no.: 111617TBSSV-2
Purity: 96.4%

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Age at first dose: 8-9 weeks
Weight range at mating (GD 0): 180-210 g
Weight range at first dose: 197.9-245.9 g
Animals were acclimated to laboratory conditions for a minimum of 1 day prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Animals were housed in one room in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information. Animals were individually housed.

Temperature: 20 to 26°C
Humidity: 30 to 70%
Light/Dark cycle:12-hour light/12-hour dark
Air changes: Minimum of 10 air changes per hour
Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum.
Water: Filtered water was provided ad libitum via an automatic watering system supplemented with water bottles as needed.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

The formulations were placed at room temperature prior to dosing. The animals were dosed daily on GD 5 to 20 (day of confirmation of mating = GD 0) via oral gavage at a volume of 2.0 mL/kg. Dose volumes were based on the most recent body weights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analysis of the dose formulations was performed as part of the method validation. Test substance formulations were solutions; therefore, homogeneity sampling and analysis was not required. Samples from dose formulations prepared for week 1 and last week were analyzed for concentration verification using a validated method.

Details on mating procedure:

Time-mated female Sprague Dawley rats were received by the test facility.

Duration of treatment / exposure:

The animals were dosed daily on GD 5 to 20 (day of confirmation of mating = GD 0).

Frequency of treatment:

Daily once

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were initially accepted into the randomization pool based upon GD 0 body weights provided by the vendor. The suitability of the randomized animals was confirmed by physical examinations upon arrival. They were assigned to study groups using computer-generated random numbers such that the mean body weight for each group was not statistically different (p ≤ 0.05) from the control mean. Following randomization each study animal was assigned a unique number and identified by an ear tag.

Maternal examinations:

Cageside observations included observation for mortality, morbundity, general health, and signs of toxicity (only abnormal findings were recorded during the cageside observations). Physical examinations included evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behavior patterns.

Ovaries and uterine content:

The abdominal cavity was opened, the uterus was excised, and the gravid uterine weight was recorded. Beginning at the distal end of the left uterine horn, the location of viable and nonviable fetuses, early and late resorptions for each uterine horn, and the number of total implantations were recorded. The number of corpora lutea on each ovary was also recorded. The embryonic membrane of each fetus was gently removed. The fetuses were removed from the uterus and the placentae were grossly examined. Each implant was categorized as viable, nonviable, late resorption, or early resorption. Uteri from females that appeared to be nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. The foci, if detected, were considered early resorptions, and data from these females were not included in mean calculations and statistics. If no foci were seen, the female was considered to be non-pregnant and data from these females were also not included in mean calculations or statistics.

Fetal examinations:

All live fetuses were individually weighed, identified, sexed, and examined for external malformations and variations. Following completion of the external examination of all fetuses in the litter, each fetus was euthanized. Approximately one-half of the fetuses in each litter were examined viscerally by fresh tissue dissection. The sex of the fetus was recorded. The heads were examined by Wilson’s sectioning. The remaining fetuses had the internal sex recorded and then were eviscerated, preserved, stained with Alizarin Red S and Alcian Blue, and examined for skeletal abnormalities. Fetal findings were classified as malformations or developmental variations. All fetal malformations were photographed, digitally saved and archived with the study file.
Intact fetuses from dams that died within 24 h of scheduled euthanasia were evaluated to the extent possible for external malformations and variations and sexed. All other intact fetuses from females not surviving to scheduled euthanasia were examined externally to the fullest possible extent and discarded.

Statistics:

See under "any other information on materials and methods incl. tables".

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

no effects observed

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significant reduction in gravid uetrine weight which corresponded to reduced body weights.

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: general systemic toxicity

Fetal body weight changes:

effects observed, treatment-related

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

LOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: Reduced body weight and reduction in body weight gain

Description (incidence and severity):

observed at all dose levels

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

20 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the study conditions, the NOAEL for maternal systemic toxicity was determined to be 20 mg/kg bw/day. No NOAEL for fetal developmental effects could be determined due to effects at all doses and therefore, the LOAEL for fetal developmental toxicity was considered to be 20 mg/kg bw/day.

Executive summary:

A prenatal developmental toxicity study was conducted in Sprague Dawley rats according to OECD Guideline 414, in compliance with GLP. The test substance was administered to groups of 25 time-mated Sprague Dawley female rats at 0, 20, 60 or 100 mg/kg bw/day through oral gavage from Gestation Day 5 to 20 (GD 5 to GD 20). Dams were subjected to a full gross necropsy and uterine and fetal examinations on GD 21. Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology examinations, uterine data, and fetal examination data (external, visceral, skeletal, and head exams). Dose concentration analysis of samples from formulations prepared for Week 1 and the last week of dosing confirmed that the test substance was properly formulated. Administration of the test substance resulted in maternal mortality at 100 mg/kg bw/day due to body weight loss, decreased food consumption and associated clinical signs of rough haircoat and thin appearance. At 60 mg/kg bw/day, the adverse effects included reduced food consumption and body weight in the dams. There were no effects on pregnancy and uterine parameters including the number of live fetuses as a percentage of post-implantation sites. There was an increase in the incidence of fetal defects (malformations and variations) in all the treated groups. These skeletal variations included incomplete ossification and unossification of the sternebrae and cervical and thoracic centrum and were attributed to the increase in the incidence of unossified or incomplete ossification. These skeletal variations were considered secondary to the reduction in fetal body weight and associated with delayed ossification. Due to their reversible nature, these variations were considered non-adverse, as the offspring would continue to grow and the ossification process would continue until complete with no expected structural alterations. Under the study conditions, the NOAEL for maternal systemic toxicity was determined to be 20 mg/kg bw/day. No NOAEL for fetal developmental effects could be determined due to effects at all doses and therefore, the LOAEL for fetal developmental toxicity was considered to be 20 mg/kg bw/day (Murphy, 2019c).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Reproductive toxicity

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats according to OECD Guideline 422, in compliance with GLP. Male rats were administered the test substance at doses of 0, 10, 30 or 100 mg/kg bw/day for 14 d prior to cohabitation and during cohabitation for at least a total of 28 d. Female rats were dosed with the test substance at doses of 0, 10, 30 or 60 mg/kg bw/day prior to cohabitation, during cohabitation, through pregnancy, and throughout lactation to Postnatal Day 13 (PND 13). Treatment-related effects in male rats included a significant effect on the body weight, body weight changes, organ weights, and microscopic findings. For female rats, repeat dose toxicity was observed at doses of 30 and 60 mg/kg bw/day primarily due to reduced body weight and microscopic findings in liver, kidney and thyroid. Important microscopic findings consisted of cytoplasmic alteration in hepatocytes at doses greater than 30 mg/kg bw/day in both sexes, thyroid follicular cell hypertrophy in males from 10 mg/kg bw/day and vacuolation of distal tubules in the kidney in males and females at the respective high doses. The test substance induced reproductive toxicity at doses of 30, 60 (females only) and 100 (males only) mg/kg bw/day. Treatment-related degeneration/atrophy of seminiferous tubules in all high dose males resulted in complete loss of fertility at this dose. Although degeneration of spermatids was only identified in one animal administered 30 mg/kg bw/day, this change was associated with decreased fertility and fecundity; therefore, testicular changes were adverse at or above 30 mg/kg bw/day. Decreases in testes and epididymal weight correlated with these changes in the high dose males. In females at 60 mg/kg bw/day, no viable pregnancy was observed and pups could not be evaluated. This was likely due to the effect on spermatogenesis, as there was evidence of copulation and normal estrus cyclicity at the time of cohabitation. The copulation index, fertility index and pregnancy rate are significantly and dose-dependently reduced due to the treatment-related effects in males. The test substance also induced developmental toxicity at 30 mg/kg bw/day evidenced by growth inhibition and reduced litter size without any indication of induced specific malformations. Therefore, the reduced pup growth and litter size could potentially be due to maternal and/or paternal toxicity rather than direct impairment of development in utero, but the potential for association between the treatment and direct impairment of in utero development cannot be ruled out based upon the results of this study. The dose group of 60 mg/kg bw/day could not be evaluated for developmental toxicity due to dams not producing any litters. Under the study conditions, the LOAEL for systemic toxicity in parental male rats was determined to be 10 mg/kg bw/day (due to microscopic findings in thyroid). The NOAEL for systemic toxicity in female rats was considered to be 10 mg/kg bw/day due to adverse body weight changes, clinical chemistry, organ weight and microscopic findings. The NOAEL for reproductive toxicity in both male and female rats was determined to be 10 mg/kg bw/day dose. The NOAEL for fetal developmental toxicity was determined to be 10 mg/kg bw/day (Murphy, 2019b).

Developmental toxicity

A prenatal developmental toxicity study was conducted in Sprague Dawley rats according to OECD Guideline 414, in compliance with GLP. The test substance was administered to groups of 25 time-mated Sprague Dawley female rats at 0, 20, 60 or 100 mg/kg bw/day through oral gavage from Gestation Day 5 to 20 (GD 5 to GD 20). Dams were subjected to a full gross necropsy and uterine and fetal examinations on GD 21. Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology examinations, uterine data, and fetal examination data (external, visceral, skeletal, and head exams). Dose concentration analysis of samples from formulations prepared for Week 1 and the last week of dosing confirmed that the test substance was properly formulated. Administration of the test substance resulted in maternal mortality at 100 mg/kg bw/day due to body weight loss, decreased food consumption and associated clinical signs of rough haircoat and thin appearance. At 60 mg/kg bw/day, the adverse effects included reduced food consumption and body weight in the dams. There were no effects on pregnancy and uterine parameters including the number of live fetuses as a percentage of post-implantation sites. There was an increase in the incidence of fetal defects (malformations and variations) in all the treated groups. These skeletal variations included incomplete ossification and unossification of the sternebrae and cervical and thoracic centrum and were attributed to the increase in the incidence of unossified or incomplete ossification. These skeletal variations were considered secondary to the reduction in fetal body weight and associated with delayed ossification. Due to their reversible nature, these variations were considered non-adverse, as the offspring would continue to grow and the ossification process would continue until complete with no expected structural alterations. Under the study conditions, the NOAEL for maternal systemic toxicity was determined to be 20 mg/kg bw/day. No NOAEL for fetal developmental effects could be determined due to effects at all doses and therefore, the LOAEL for fetal developmental toxicity was considered to be 20 mg/kg bw/day (Murphy, 2019c).

Mode of Action Analysis / Human Relevance Framework

Due to the findings in offspring, no specific Mode of Action could be identified.

The findings could be relevant for humans.

Justification for classification or non-classification

Based on the results of combined repeated dose toxicity with reproduction-developmental toxicity screening test, subchronic repeated dose toxicity testing and the pre-natal developmental toxicity studies in rats, the test substance warrants classification as Rep. Cat. 2 (H361f: May damage fertility) according to CLP (1272/2008/EC) criteria. The available information is not sufficient for classification as Rep. Cat. 1, as a partial reversal of effects were seen in the subchronic repeated dose toxicity testing and low to mid exposure within the tests did not result in severe effects.