S-2-Benzothiazolyl (Z)-2-(2-aminothiazol-4-yl)-2-acetyloxyiminothioacetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Conclusion from the OECD421 study with rats and oral administration of the test substance:

Effects of the test substance on parental body weight, food consumption and delivery data of dams were observed at 1000 mg/kg bw/d.

Effects on F1 were reduced body weight and a shorter anogenital distance in female pups at 1000 mg/kg bw/d.

NOAEL for systemic toxicity of male/female rats: 300 mg/kg bw/day

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

NOAEL for reproductive performance (based on delivery data of dams): 300 mg/kg bw/day

NOAEL for F1 Offspring: 300 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:

Start of treatment: January 2017. Necropsy: February/March 2017. Final report: February 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2000

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

According to the guideline OECD421.

Specific details on test material used for the study:

Test item name: t15-AE
Batch No.: 31139692
Chemical name: S-2-Benzothiazolyl (Z)-2-(2-aminothiazol-4-yl)-2-acetyloxyiminothioacetate

Species:

rat

Strain:

other: Hsd.Han: of Wistar origin

Details on species / strain selection:

The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
Age of animals at start of the study: Male animals: 85 – 87 days; Female animals: 97 – 100 days
Body weights at start of the study: 347 – 422 g for male animals; 215 – 249 g for female animals
The weight variation did not exceed ± 20 per cent of the mean weight
Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder.
Acclimatization time: 27 days

Housing conditions
Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: 2 animals/ cage
Cage type: Type III polypropylene/polycarbonate;
Size: 22 x 32 x 19 cm (width x length x height)
Bedding: Certified laboratory wood bedding (Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg). The bedding is suitable as nesting material. Details of quality of bedding material were reported.
The cages and bedding were changed twice a week.
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: Above 10 air-exchanges/ hour by a central air-condition system.
Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum. Food and water quality was checked.
Animals were identified by unique numbers.

Route of administration:

oral: gavage

Vehicle:

other: 1 % methylcellulose

Details on exposure:

Vehicle: Methylcellulosum Ph.Eur 7. Batch number: AX4380542. In destilled water. A treatment volume of 5 mL/kg body weight was applied.
t15-AE was formulated in the vehicle in concentrations of 200, 60, and 20 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days and stored in a refrigerator (at 5 ± 3 °C) until use.

Details on mating procedure:

Mating begun 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage. Females remained with the same male until copulation occurred.
Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (control of concentration) was performed twice during the study. Five aliquots of 5 mL of each formulation (200, 60 and 20 mg/mL) and five aliquots of 5 mL control substance (vehicle) were taken and analyzed.
Concentration of the test item in the dosing formulations varied in the range of 93 and 107% of the nominal values at both analytical occasions.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 97 % at ca. 1 mg/mL and 100 % at ca. 200 mg/mL).
t15-AE proved to be stable in a refrigerator (at 5 ± 3 °C) for three days and at room temperature for one day. A separate analytical report (Study no. 880-100-1656) provided these data, see Section 8, Kiss2016.

Duration of treatment / exposure:

The experimental period involved 27 days of acclimatization (including 14 days for examination of estrous cycle) and 55 days treatment/observation period and necropsy day. The day of first treatment was considered as Day 0 of examination.

Frequency of treatment:

Once a day.

Details on study schedule:

Males:
Acclimatization period 27 days -> Treatment starts -> Pre-mating period 14 days -> Mating period 1 – 5 days -> Post mating period 36 – 40 days -> Necropsy, organ weighing Day 55.
Blood sampling for T4 examinations on postpartum day 13.

Females:
Acclimatization period including examination of estrous cycle: 27 days -> Treatment starts -> Pre-mating period 14 days -> Mating period 1 – 5 days -> Gestation period 22-23 days -> Deliver -> Lactation period post partum (PP) 14 – 18 days -> Necropsy dams PP days 15 – 19.
Blood sampling for T4 examinations on postpartum day 13.

Dosing of both sexes begun after 27 days acclimatization and was continued up to and including the day before the necropsy.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Number of animals involved in the study: 48 males, 48 females (nulliparous, non-pregnant females)
Number of animals/group: 12 animals/sex in the control and dose groups (at least 8 pregnant female animals per group were expected)
Number of groups: 4 (3 dose levels + 1 control group)

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen on the basis of the results of a preliminary toxicity screening test with t15-AE in rats, see Section 7.5.1 Szakonyine 2017.

Positive control:

No.

Parental animals: Observations and examinations:

Inspection for signs of morbidity and mortality twice daily.
General clinical observations were made on parental animals once a day.
Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum.
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also determined by weekly interval during post-mating period.
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If negative on day 13, the examination was repeated on day 14 of gestation.
Blood samples for assessment of serum thyroid hormones (T4) were collected from all parental male and female animals.

Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. Dams were observed whether they made a nest from the bedding material and nurse their new-born or not. The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
On post-partum day 4, the size of each litter was adjusted to four pups per sex per litter, if feasible. Extra pups were eliminated by a random selection. Partial adjustment of litter size was performed if the number of male and female pups did not allow having four of each sex per litter (for example, five males and three females).

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.
Each morning a vaginal smear were prepared and stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope.

Sperm parameters (parental animals):

Histopathology examinations were performed on testes, epididymides of the animals in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

Each litter were examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition is complete, day 0), and on days 4 and 13 post-partum with an accuracy of 0.1 g. Any abnormal behavior of the offspring was recorded.
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups).
All litters were checked and recorded daily for the number of viable and dead pups. Dead pups found were subjected to necropsy by macroscopic examination. All observed abnormalities were recorded.
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore individual body weight of pups was also determined on postnatal day 4.
The number of nipples/areolae in male pups was counted on postnatal day 13.

Blood samples for serum T4 assessment were pooled from at least two pups per litter on postnatal day 4 (if it was feasible) and on postnatal day 13.

Postmortem examinations (parental animals):

The ovaries, uterus with cervix and fallopian tube, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands of all adult animals were preserved.
Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.

At the time of termination, body weight, brain weight and weight of the testes and epididymides, prostate and seminal vesicles with coagulating gland (as a whole) of adult animals were determined.

Full histopathology examinations were performed on the ovaries, testes, epididymides of the animals in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.

Reproductive indices:

Males:
− Percentage of pairings
− Percentage of fertile males
− Percentage of infertile males
− Male copulatory index
− Male fertility index
Females:
− Percentage of pairings /sperm positive females
− Percentage of pregnant females
− Percentage of non-mated females
− Female copulatory index
− Female fertility index
− Gestation index
− Duration of pregnancy (days)
− Number of implantations / dams
− Post-implantation mortality
− Number of dams with live pups on lactation days 0, 4 and 13

Offspring viability indices:

− Number of live births per litter
− Number of viable pups per litter on postnatal days 0 and 13
− Post-natal mortality /Extra-uterine mortality
− Survival Index of pups on postnatal day 13
− Sex ratio % (on postnatal days 0 and 13)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations. At the same intervals, the behavior and physical condition of the animals was not impaired at each dose level (1000, 300 or 100 mg/kg bw/day) during the entire treatment period. Two dams at 1000 mg/kg bw/day showed clinical signs (piloerection, paleness or decreased body tone) before or during the parturition

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality at any dose level (1000, 300 and 100 mg/kg bw/day).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight and body weight gain were reduced at 300 and 1000 mg/kg bw/day in male animals during the entire treatment period. The difference in the mean body weight was lower than 10 % with respect to the control group at 300 mg/kg bw/day.
The mean body weight gain of male animals at 100 mg/kg bw/day was comparable to the control during the entire treatment period.
The body weight of female animals was also reduced with respect to the control at 1000 mg/kg bw/day during the gestation and lactation periods, which however may be due to the lower number of pups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption was slightly reduced with respect to the control in male animals at 300 and 1000 mg/kg bw/day in accordance with the body weight changes during the pre-mating period. In the female animals at 300 and 1000 mg/kg bw/day, the food consumption was transiently reduced during week 1. Slight changes with respect to the control at the end of gestation period (1000 and 300 mg/kg bw/day) at and during the lactation period (at 1000 mg/kg bw/day) was probably due to the lower number of pups.

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

Description (incidence and severity):

The serum T4 levels were not affected in parental male animals or in PN 13 offspring in the test item administered groups comparing to their control.

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):

Histopathological examinations of the selected organs (ovaries, testes, epididymides,) did not reveal any test item related changes at any dose level.
In the male animals the investigated organs of reproductive system (testes and epididymides) were histologically normal and characteristic on the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides was normal in all cases, as well.
In the female animals the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.
In one control male animal, subacute corneitis accompanied with vascularization (right side) was detected. No histological lesions were observed in the left side eye. The other parts of the affected eye (iris, lens, retina, ciliary body, choroid) were normal. Inflammation of the cornea occurs occasionally and may be secondary to trauma or other causes (bacterial or viral infection). In this case – based on the one side occurrence and the histological character of the lesion – it was considered as mechanical origin.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The percentage of female animals with regular cycle was lower at 1000 mg/kg bw/day with respect to the control resulting in statistically significant differences in the examined parameters of estrous cycle. A test item influence on the estrous cycle cannot be verified or excluded by the results of this study as these slight changes in the estrous cycle had no influence on female reproductive performance at 1000 mg/kg bw/day.
See also the attachment.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were no test item related differences between the control and test item treated groups in the gonadal function, mating behavior, conception, pregnancy, parturition of male or female animals (1000, 300 and 100 mg/kg bw/day).
The mean number of implantation, total birth and liveborns and alive pups were significantly lower in 1000 mg/kg bw/day while the pre-natal loss as well as total loss was higher at 1000 mg/kg bw/day than in the control group.
See also the attachment.

Dose descriptor:

NOAEL

Effect level:

300 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

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

other: No specific organ toxicity was observed, only effects on body weight and food consumption, and on delivery data of dams.

Organ:

other: No specific organ toxicity was observed, only effects on body weight and food consumption, and on delivery data of dams.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
One pup of a control dam has filiform tail as an individual alteration.
The percentage of cold offspring was higher in the control group then in high dose group groups than in the control group.
Absence of milk in the stomach was noted with higher incidence in the test item treated groups. In the lack of any dose relevancy and because of the transient occurrence (it was mainly detected on the day of birth) it was considered to be not related to the test item.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.
The mean number of dead and missing offspring per litter was similar in all groups. There were no significant differences between the control and test item treated groups (1000, 300 or 100 mg/kg bw/day) in the survival indices on postnatal days 4 or 13.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test item related effect on the body weight development of the offspring was observed at 1000 mg/kg bw/day.
The mean litter weight was significantly lower than in the control group at 1000 and 300 mg/kg bw/day at the birth and on postnatal day 4, and at 1000 mg/kg bw/day on postnatal day 13. The litter weight gain was also below the control value at 1000 and 300 mg/kg bw/day between postnatal days 0 and 4 and at 1000 mg/kg bw/day between postnatal days 4 and 13 as well as for the whole observation period (between postnatal days 0 and 13).
The mean pup weight was also lower than in the control group at 1000 mg/kg bw/day on postnatal days 4 and 13. Similarly, the mean body weight gain of pups was lower with respect to their control between postnatal days 0 and 4 and between postnatal days 0 and 13.
The changes in litter weight at 300 mg/kg bw/day were slight and were not considered toxicologically relevant as there were no significant differences in the mean pup weight and weight gain. More specifically, the mean pup weight and the mean weight gain of pups were similar in the control and 300 mg/kg bw/day groups on postnatal days 0, 4 and 13 as well as between days 0 and 4, between days 0 and 13. The mean body weight gain of pups at 300 mg/kg bw/day was higher than in the control group between postnatal days 4 and 13.

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

Description (incidence and severity):

The serum T4 levels were not affected in PN 13 offspring in the test item administered groups comparing to their control.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
Autolyzed visceral organs were seen in one stillborn at 1000 mg/kg bw/day (1/2) on the day of birth, in dead pups in control (1/2) and in 300 mg/kg bw/day (1/2) groups on post-natal day 1. One dead pup at 1000 mg/kg bw/day (1/2) was pale and without macroscopic visceral alteration.
The stomach was gas filled (no milk) in one dead pup at 100 mg/kg bw/day (1/2). One pup at 300 mg/kg bw/day (1/2) was cannibalized.
There were no macroscopic findings in stillborns of control (1/2) and in 100 mg/kg bw/day (1/2) groups on post-natal day 0.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Sex Distribution
There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on postnatal days 0, 4 or 13.
Statistically significant difference with respect to the control was noted for the lower mean number of offspring per litter at 1000 mg/kg bw/day on postnatal days 0, 4 and 13 and for the lower mean number of male pups on postnatal day 0 as well as for the lower mean number of offspring at 300 mg/kg bw/day on postnatal day 4. There were no significant differences in the percentages of genders between the control and test item treated groups during the observation period.
See also the attachment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Development of offspring
The anogenital distances (absolute and normalized) were slightly shorter than in the control in female pups at 1000 mg/kg bw/day.
Statistical significance was noted for the shorter absolute anogenital distance of male pups with respect to the control. The normalized anogenital distance of male pups was similar in the control and 1000 mg/kg bw/day.
The anogenital distance (absolute) was shorter in the female pups of dosed groups (1000, 300 and 100 mg/kg bw/day) with respect to the control. The normalized anogenital distance was slightly shorter than in the control in female pups at 1000 mg/kg bw/day.
Nipples/areoles were not visible in any of the examined male offspring in the control or 1000, 300 or 100 mg/kg bw/day groups on postnatal day 13.
See also the attachment.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

other: No specific organ toxicity was observed, only effects on body weight and on normalized anogenital distance of female pups.

Organ:

other: No specific organ toxicity was observed, only effects on body weight and on normalized anogenital distance of female pups.

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Regarding the relation to other toxic effects: Reproductive effects are occurring together with other (non-specific) maternal toxic effects, therefore not unlikely occurring as a secondary non-specific consequence of other toxic effects. This can not be expressed in the Table above.

Conclusions:

Effects of the test substance on parental body weight, food consumption and delivery data of dams were observed at 1000 mg/kg bw/d.
Effects on F1 were reduced body weight and a shorter anogenital distance in female pups at 1000 mg/kg bw/d.

Executive summary:

An investigation according to the OECD Guidelines 421 “Reproduction / Developmental Toxicity Screening Test” was performed with Wistar rats and oral administration. Doses used: 0 - 100 - 300 - 1000 mg/kg bw/d. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 55 days). Females were additionally exposed through the gestation period and up to lactation days 14 - 18, i.e. up to the day before necropsy (altogether for 55 days).

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. Blood samples were collected for possible determination of serum levels of thyroid hormones (T4) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/ post-natal day 13 and from all parent male animals and from one non-pregnant female at termination (Day 55).

The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides of adult male animals were determined. Thyroid gland was preserved from all adult males and females and one male and one female pup per litter (at day 13) for the intended subsequent histopathological examination.

Results:

Reduced body weight development and food consumption were observed at 1000 mg/kg bw/day. In female animals at 1000mg/kg bw/day, the changes in body weight and food consumption at the end of the gestation period and during the lactation period may be related to the lower number of offspring. At 300 mg/kg bw/day, slight and not considered to be toxicologically relevant changes in body weight, body weight gain and food consumption were (male) were detected. There were no test item related alterations in the examined parameters at 100 mg/kg bw/day (male and female).

The reproductive performance (gonad function, mating behavior, conception, parturition) of parental males and females was not affected at 1000, 300 or 100 mg/kg bw/day. The copulatory and fertility indices were comparable in the control and test item administered animals (male and female). The delivery data of dams were affected at 1000 mg/kg bw/day (higher pre-natal loss, lower mean number of implantation, total birth and liveborns and alive pups with respect to the control).

Effects on development of offspring development were observed at 1000 mg/kg bw/day as the mean litter weight and weight gain as well as the pup weight and weight gain were reduced with respect to the control. The normalized anogenital distance of female pups was slightly shorter than in the control group.

NOAEL for systemic toxicity of male/female rats: 300 mg/kg bw/day

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

NOAEL for reproductive performance (based on delivery data of dams): 300 mg/kg bw/day

NOAEL for F1 Offspring: 300 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Mode of Action Analysis / Human Relevance Framework

No (reproductive) organ toxicity, but effects of the test substance on parental body weight, food consumption and delivery data of dams at 1000 mg/kg bw/d, as well as reduced body weight and a shorter anogenital distance in female pups at 1000 mg/kg bw/d in the F1 generation were observed. No specific mode of action is derived. Reproductive effects are occurring together with other toxic effects, possibly as a secondary non-specific consequence of the other toxic effects.

The human relevance is not specified.

Justification for classification or non-classification

Conclusive but not sufficient for classification.

Reproductive effects were observed together with other maternal toxic effects at the high dose of 1000 mg/kg bw/d, therefore not unlikely occurring as a secondary non-specific consequence of other toxic effects. No classification is therefore derived.