(6S,7S)-7-Amino-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of treatment: April 2017. Treatment up to June 2017. Final report: February 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

not specified

Test guideline 2

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.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

435-680-1

EC Name:

-

Cas Number:

24701-69-7

Molecular formula:

C9H12N2O4S (Hill formula) C9H12N2O4S (CAS formula)

IUPAC Name:

(6R,7R)-7-amino-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

Test item: 7-AMCA
Chemical name: 5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-amino-3-(methoxymethyl)-8-oxo, (6R,7R)-
Batch No.: B345118
CAS No.: 24701-69-7
Other components: water
Appearance: Light brown, weakly odorous powder
Manufacturing date: 01 February 2016
Retest date: 31 January 2017
Storage: Refrigerator (2-8ºC), protected from light

Specific details on test material used for the study:

Test item: 7-AMCA
Batch No.: B345118
Chemical name: 5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-amino-3-(methoxymethyl)-8-oxo-, (6R, 7R)-
Active ingredient content: ca. 92.0 %
Manufacturing date: 01 February 2016
Expiry date: January 31, 2018
Storage condition: at 5 ± 3 °C, protected from light
Appearance: Light brown, weakly odorous powder

Test animals

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: 354 – 464 g for male animals; 213 – 264 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: 35 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.

Administration / exposure

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.
The test substance 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. Two females, which failed to mate within 14 days, was mated with proven males of the same group 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 95 and 106 % 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: 108 % at ca. 1 mg/mL and 102 % at ca. 200 mg/mL).
The test substance 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. 880-100-1651) provided these data, see Section 8, Kiss 2016.

Duration of treatment / exposure:

The experimental period involved 35 days of acclimatization (including 16 days for examination of estrous cycle) and 54, 55, 62 and 67 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. 7 Days a week.

Details on study schedule:

Males:
Acclimatization period 35 days -> Treatment starts -> Pre-mating period 14 days -> Mating period 1 – 18 days -> Post mating period 22 – 39 days -> Necropsy, organ weighing Day 54.
Blood sampling for T4 examinations on postpartum day 13.

Females:
Acclimatization period including examination of estrous cycle: 35 days -> Treatment starts -> Pre-mating period 14 days -> Mating period 1 – 18 days -> Gestation period 21-22 days -> Deliver -> Lactation period post partum (PP) 14 – 18 days -> Necropsy dams: Day 54, 55, 62 or 67.
Blood sampling for T4 examinations on postpartum day 13.

Dosing of both sexes begun after 35 days acclimatization and was continued up to and including the day before the necropsy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

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 the test substance in rats.

Positive control:

No.

Examinations

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 (premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).
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 started from each animal was being considered for study for 16 days. Animals exhibited typical 4-5 days cycles were included in the study preferably. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.
Vaginal smear also was prepared on the day of the necropsy.
The vaginal smears were 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):

Gross necropsy was performed on each animal.
- Parental male animals: Day 54;
- Non-pregnant female animal: Day 54;
- Dams on post-partum day 14–18;
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, epididymides and prostate and seminal vesicles with coagulating gland (as a whole) of adult animals were determined.

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.
In addition, these organs were examined histologically in one non-pregnant female animal (no. 331 at 300 mg/kg bw/day) and male cohabited with (no. 311) and in not mated males as well (no. 206 at 100 mg/kg bw/day and no 310 at 300 mg/kg bw/day).
In the 300 mg/kg bw/day the seminal vesicle with coagulating gland of one male animal (no. 302) was examined as well as the ovary of one female (no. 329) based on macroscopic findings.
The uterus was also examined histologically in female animals showing macroscopic findings in uterus (3/12 control; 1/11 at 300 mg/kg bw/day; 1/12 at 1000 mg/kg bw/day).
Additionally, caecum from all animals was processed and histologically examined because of necropsy findings (dilatation of the caecum).

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 sperm positive, but non-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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
The behavior and physical condition of the animals was not impaired except for a few observations that were considered to be individual signs and not related to the test item.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

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

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not adversely affected by the test item in male or female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period.
The mean body weight gain was statistically significantly lower than in the control group in male animals at 1000 mg/kg bw/day between Days 7 and 13 and between Days 34 and 41. Statistical significance was also detected at the slightly higher mean body weight gain of male animals at 1000 mg/kg bw/day between Days 41 and 48.
The mean body weight and body weight gain of female animals administered with 100, 300 or 1000 mg/kg bw/day were comparable with the control during the pre-mating, gestation and lactation periods.
Statistical significance was detected at the slightly higher mean body weight gain of female animals at 100 and 300 mg/kg bw/day between gestation days 0 and 7 and at the lower mean body weight gain of females at 300 mg/kg bw/day between gestation days 14 and 21.
See also the attachment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant differences with respect to their control in the mean daily food consumption in male or female animals at 100, 300 or 1000 mg/kg bw/day during the entire observation period.
In male animals at 300 and 1000 mg/kg bw/day the mean daily food consumption was statistically significantly lower than in the control group during the first week of the pre-mating period.
Similarly, the mean daily food consumption was statistically significantly lower than in the control group in female animals at 100, 300 and 1000 mg/kg bw/day during the first week of the pre-mating period.
After the first week no significant differences were observed between the control and the test item treated groups in male and female animals, therefore these transient differences were judged to be toxicologically not relevant.

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 similar in parental male animals and PN 13 offspring in the control and test item administered groups.

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 did not reveal any test item related alterations in the selected organs or tissues of male and female animals (testes, epididymides and ovaries) at the highest dose (1000 mg/kg/bw/day). However, dilatation of caecum in several animals belonging to the treated groups was observed. No pathological lesion in the mucous membrane of affected part of intestine could be in connection with the effect of test items.

See also the attachment.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A test item influence on the estrous cycle was not detected at any dose level (100, 300 and 1000 mg/kg bw/day).
During the pre-treatment period there were no differences between the control and treated groups (100, 300 and 1000 mg/kg bw/day) in the following examined parameters of estrous cycle: the mean number or length of cycles, mean number of days in pro-estrous, estrous or diestrous during the pre-mating period, mean number of animals in prolonged or estrous diestrous. Statistically significantly lower number or percentage of animals with regular cycles was observed in the 100, 300 and 1000 mg/kg bw/day groups when compared to the control.
In the pre-mating period statistically significant differences were observed when compared to their control in the following parameters:
- higher number or percentage of animals with regular cycles at 100 mg/kg bw/day;
- higher mean number of cycles at 100 and 1000 mg/kg bw/day;
- higher mean number of days in estrous at 100 and 1000 mg/kg bw/day;
These differences of the three parameters are the consequence of the fact that in the control group relatively high number of females with irregular cycle was observed (5/12) and each of these females were in prolonged diestrous. Therefore, these differences were not considered to be toxicologically relevant.

Delivery Data of Dams
The delivery data of dams were not affected by the treatment with the test item at 100, 300 or 1000 mg/kg bw/day.
The mean number of implantation sites, pre-natal loss and postnatal loss on the day of delivery were similar in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the mean duration of pregnancy, in the mean number of total births, live-borns, stillborns or viable pups on postpartum day 0, in the percentage of dams with viable offspring on postpartum day 0 or in the live birth index.

See also the attachment.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not affected by the treatment with the test item at 100, 300 or 1000 mg/kg bw/day in male or female animals.
Although statistical significance with respect to the relevant control was noted for the slightly lower copulatory index in male animals at 100 and 300 mg/kg bw/day (one male in each group did not mated) and for the lower fertility index in male and female animals at 300 mg/kg bw/day (one pair in the group did mate, however the female was not pregnant), these slight differences were considered to be toxicologically or biologically not significant and not related to the test item.
See also the attachment.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

other: Dilatation of the caecum without any histopathological alterations was detected and is considered to be a non-adverse effect of the test item treatment and might be a physiological phenomenon.

Organ:

intestine

Results: F1 generation

General toxicity (F1)

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.
The percentage of cold and not suckled offspring was similar in the control and high dose groups. Some clinical signs – pale, wasted, smaller than normal size and cyanotic body – were only observed in pups of low and mid dose groups (except one smaller than normal control pup).

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 live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were no significant differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices. There were no significant differences between the control and test item treated groups (100, 300 or 1000 mg/kg bw/day) in the mean number of dead offspring (including missing pups) per litter. The highest total dead pup (including missing) number was observed in the mid dose (11 pups), while only one dead pup occurred in the control or in the high dose.
See also the attachment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A test item related effect on the body weight development of the offspring was not found.
The mean litter weights and the litter weight gain were similar in the control and in all test item treated groups (100, 300 and 1000 mg/kg bw/day) on postnatal days 0, 4 and 13.
Statistically significant difference with respect to the control was detected at 1000 mg/kg bw/day in the mean body weight of pups on postnatal day 0 however did not cause statistically significant difference in the mean body weight gain on this day. This difference was transient and was not detected on post-natal days 4 or 13, therefore it was not considered to have toxicological relevance.

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.
There were no macroscopic changes in the organs or tissues of the stillborn offsprings in the 100 mg/kg bw/day group (2/2) subjected to necropsy on postnatal day 0. There were no macroscopic changes in two dead pups (2/3) of this group on postnatal day 0, however one of them was smaller than normal and the other had milk in the stomach. Autolyzed visceral organs were noted for one dead pup (1/3) on post-natal day 1 in this group.
In the 300 mg/kg bw/day group there were no macroscopic changes in the organs or tissues of the stillborn offsprings (5/5) subjected to necropsy on postnatal day 0. The stomach was empty (no milk) in two dead pups (2/4) in this group subjected to necropsy on postnatal day 1.
There were no macroscopic changes in one dead offspring in this group (1/4) subjected to necropsy on postnatal day 0, however it was uncleaned and its umbilical cord was intact. There were no macroscopic changes in one dead pup (1/4) of this group on postnatal day 0.
At 1000 mg/kg bw/day no macroscopic changes in the organs or tissues of the stillborn offspring (1/1) subjected to necropsy on postnatal day 0 was observed. One pup of this group was partially cannibalized (1/1) on post-natal day 0, performance of lung flotation test and sex determination was not feasible in this case. Autolyzed visceral organs were noted for one dead pup (1/1) on post-natal day 1 in this group.

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.
See also the attachment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Development of offspring
The anogenital distances (absolute and normalized in male or female offspring) or nipple retention (male) were not affected by the test item treatment at 100, 300 and 1000 mg/kg bw/day.
The mean absolute anogenital distance was statistically significantly shorter in male pups of the 100 and 1000 mg/kg bw/day as well as of the mean normalized anogenital distance of 1000 mg/kg bw/day.
Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on postnatal day 13.
The mean body weight of male and female offspring at 1000 mg/kg bw/day was slightly but statistically significantly lower than in the control group on postnatal day 4.
See also the attachment.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

Target system / organ toxicity (F1)

Critical effects observed:

no

System:

other: No specific organ toxicity was observed.

Organ:

other: No specific organ toxicity was observed.

Overall reproductive toxicity

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Effects of the test substance on the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) were not observed. The test substance did cause adverse systemic toxicity in parental male and female rats.
The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13.

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 54 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 17, i.e. up to the day before necropsy (altogether for 54, 55, 62 or 67 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 16 days 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 54).

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:

Mortality: There was no test item related mortality at any dose level in males of females.

Clinical observation: 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 during the entire treatment period.

Body weight and body weight gain: The body weight development of male and female animals was not disturbed and it was comparable in the control and test item treated groups.

Food consumption: The mean daily food consumption was not adversely affected by the test item in male or female animals. The mean daily food consumption was slightly transitionally reduced with respect to the control in male and female animals at 100, 300 and 1000 mg/kg bw/day in the first week of the treatment period.

Estrous cycle: There was no test item effect on the estrous cycle of female animals.

Reproductive performance: 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.

There were no test item related differences between the control and test item treated groups in delivery data of dams.

Clinical biochemistry: The serum T4 levels were not influenced in parental male animals or in PN 13 offspring in test item administered groups comparing to their control.

Necropsy: Macroscopic findings (dilatation of the caecum) related to the test item treatment were found in male and female animals in the low dose (1/12 male, 4/12 female), mid dose (3/12 male, 11/12 female) and high dose (12/12 male and 12/12 female). The incidence of the observation increased in a dose-dependent manner.

Organ weight: There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides, prostate, seminal vesicles with coagulating gland as a whole of male animals at any dose level.

Histopathology: Histopathological examinations of the selected organs (ovaries, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related adverse changes at any dose level. However, dilatation of caecum related to the test item treatment in a proportion of animals belonging to the treated groups without pathological lesion was observed in the test item treated groups.

Offspring: The offspring’s development was not influenced at any dose level. No effect on mortality, clinical signs, body weight or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Conclusion

The test substance at 100, 300 or 1000 mg/kg bw/day did not adversely influence the reproductive performance and did not cause adverse systemic toxicity in parental male and female rats. However macroscopic findings (dilatation of the caecum) related to the test item treatment were found in male and female animals in each test item treated groups which incidence increased dose-dependently. In the lack of any histopathological alterations in the caecum this macroscopic observation is considered to be a non-adverse effect of the test item treatment and might be a physiological phenomenon.

The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

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

NOAEL for F1 Offspring: 1000 mg/kg bw/day