Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of treatment: January 2017. Necropsy: March 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
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Dose range finding study.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
yes
Remarks:
Only the dose range finding part is reported.
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
yes
Remarks:
Only the dose range finding part is reported.
Qualifier:
equivalent or similar to guideline
Guideline:
other: OPPTS 870.3050
Version / remarks:
2000
Deviations:
yes
Remarks:
Only the dose range finding part is reported.
Principles of method if other than guideline:
The dose range finding part of a subacute toxicity study is reported.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
methylcellulose
Details on oral exposure:
The test item was suspended in the vehicle (1 % methylcellulose) in concentrations of 20, 60 and 200 mg/mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations was performed once during this study. Five aliquots of 5 mL of each formulation to be administered to the animals (20, 60 and 200 mg/mL) and five aliquots of 5 mL control substance (vehicle) were taken.
The test item concentrations in the samples varied within the range from 90 % to 92 % in comparison to the nominal values.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily once.
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:
5 m + 5 f per group
Control animals:
yes
yes, concurrent vehicle
Positive control:
No.
Observations and examinations performed and frequency:
Examinations performed:
Mortality.
Clinical observations.
Body weight.
Food consumption.
Hematology ablood coagulation.
Clinical chemistry.
Sacrifice and pathology:
Gross pathology and organ weight determinations.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight gain was reduced during the course of the study resulting in lower mean body weight with respect to the control in male animals at 300 and 1000 mg/kg bw/day from Day 7 up to the end of the study. The difference in body weight with respect to the control group was lower than 10 % therefore this minor change in the body weight development was not considered to be adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
mortality
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Conclusions:
No toxicologically significant changes in the examined parameters was observed after the 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day doses.
Executive summary:

No toxicologically significant changes in the examined parameters was observed after the 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day doses.

Based on the observations made in this toxicity study the dose levels for a Reproduction/ Developmental Toxicity Screening Test in the Rat (main study) were determined as follows:

Group 1 Vehicle control

Group 2 100 mg/kg bw/day

Group 3 300 mg/kg bw/day

Group 4 1000 mg/kg bw/day

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
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.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
477-080-2
EC Name:
-
Cas Number:
103121-85-3
Molecular formula:
C13 H19 N3 O3 S . HCl (Hill Formula) C13 H20 N3 O3 S . Cl (CAS Formula)
IUPAC Name:
477-080-2
Test material form:
solid

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: 91-96 days; Female animals: 97-101 days
Body weights at start of the study: 349 – 416 g for male animals; 204 – 263 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: 41 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 four 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. Mating pair was changed at one control female animal (no. 131) on 15th day of mating to ensure the proper number of pregnant control females.
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 3 times 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 formulations at 20 and 60 mg/mL was 84 and 87 % of the nominal concentrations at the first analytical occasion (January 25, 2017) for unknown reasons. Therefore first sampling was repeated on February 01, 2017. The results of the second and third analysis met the acceptance criteria (within ± 10 % of the nominal concentrations) therefore results of first analysis were disregarded.
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: 98 % at ca. 1 mg/mL and 103 % at ca. 200 mg/mL).
The test substance proved to be stable in a refrigerator (at 5 ± 3 °C) for 4 days and at room temperature for one day. A separate analytical report provided these data.
Duration of treatment / exposure:
The experimental period involved 41days of acclimatization (including 14 days for examination of estrous cycle) and 55 / 64 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 41 days -> Pre-mating period 14 days -> Mating period 1 – 16 days -> Post mating period 25 – 40 days -> Necropsy, organ weighing Day 55.
Blood sampling for T4 examinations on postpartum day 13.

Females:
Acclimatization period including examination of estrous cycle: 41 days -> Pre-mating period 14 days -> Mating period 1 – 16 days -> Gestation period 22 days -> Deliver -> Lactation period post partum (PP) 12 – 18 -> Necropsy dams PP 13 – 19.
Blood sampling for T4 examinations on postpartum day 13.

Dosing of both sexes begun after 41 days acclimatization and was continued up to and including the day before the necropsy.
Doses / concentrationsopen allclose all
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 the test substance in rats, see Section 7.5.
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 (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.

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 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 at the detailed weekly clinical observations.
There were no test item related clinical signs in any group, i.e. the parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities at 100, 300 or 1000 mg/kg bw/day. Alopecia, a common spontaneous finding in this strain of experimental rats, was observed and is seen also in untreated rats.
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.
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, non-treatment-related
Description (incidence and severity):
The body weight development was not adversely affected in male or female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period.
There were no significant changes in the mean body weight, therefore minor changes in body weight gain of male animals were considered to be toxicologically not relevant.
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.
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.
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):
effects observed, non-treatment-related
Description (incidence and severity):
See above under Clinical Signs.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
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).
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 (storage of mature spermatozoa) was normal in all cases, as well (12/12 control; 1/1 at 100 mg/kg bw/day; 2/2 at 300 g/kg bw/day; 12/12 at 1000 mg/kg bw/day).
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 (dams: 12/12 control; 11/11 at 1000 mg/kg bw/day; non-pregnant female: 1/1 at 100 mg/kg bw/day, 2/2 at 300 g/kg bw/day, 1/1 at 1000 mg/kg bw/day).
Myoma in the uterine horns was observed in one dam at 300 mg/kg bw/day (1/10) and in another one at 1000 mg/kg bw/day (1/11), which was considered as incidental finding without toxicological significance.
In some cases dilatation of uterine horns was observed (1/12 control dam and 1/1 dam at 300 mg/kg bw/day; 1/1 non-pregnant female animal at 100 mg/kg bw/day) without other pathological findings and was considered to be a slight neuro-hormonal phenomenon in connection with the sexual function – proestrus phase – in the inner genital organs.
See also the attachment.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
A test item influence on the estrous cycle was not detected at any dose level (1000, 300 and 100 mg/kg bw/day).
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 1000, 300 or 100 mg/kg bw/day in male or female animals.
Although statistical significance with respect to the relevant control was noted for the slightly higher copulatory index in male animals at 100, 300 and 1000 mg/kg bw/day and for the lower fertility index in male and female animals at 100, 300 and 1000 mg/kg bw/day, these slight differences were considered to be toxicologically or biologically not significant and not related to the test item.
The copulatory index, percentage of pregnant females, dams delivered, pregnants not delivered, pregnants with liveborn(s) and conceiving days were comparable in female animals of control and test item treated animals.
See also the attachment.

Details on results (P0)

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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
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
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
other: No relevant effects were observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
other: No relevant effects were observed.

Target system / organ toxicity (P0)

Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: No specific organ toxicity was observed. No relevant effects were observed.
Organ:
other: No specific organ toxicity was observed.

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.
There were no clinical signs in offspring of high dose group.
The percentage of cold and not suckled offspring was slightly higher in the control group then in high dose group.
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. The mean number of live pups per litter, the mean number of viable pups per litter on postnatal days 0, 4 and 13 were similar in all groups.
There were no differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices on postnatal days 4 or 13.
See also the attachment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A test item related effect on the body weight development of the offspring was not found.
See also the attachment.
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
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.
The stomach was empty (no milk) and intestines were gas filled in one dead pup in the control group subjected to necropsy on postnatal day 7 (1/1).
There were no macroscopic findings in stillborn pups of control (7/7) and of 100 mg/kg bw/day (1/1) groups on postnatal day 0.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
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.
There were no significant differences in the percentages of genders between the control and test item treated groups during the observation period.

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) were similar in the control and test item treated groups (1000, 300 and 100 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 anogenital distance (absolute) was slightly shorter than in the control in male pups at 300 mg/kg bw/day probably along with the slightly lower mean body weight of these pups. There was no difference with respect to their control at the normalized anogenital distance of this group.
The mean body weight of female offspring was slightly but statistically significantly higher than in the control group in female offspring at 100 mg/kg bw/day.
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:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
other: No relevant effects were observed.

Target system / organ toxicity (F1)

Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: No specific organ toxicity was observed. No other relevant effects were observed.
Organ:
other: No specific organ toxicity was observed.

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
The test substance did not cause signs of systemic toxicity and did not adversely influence the reproductive performance in rats.
The development of the F1 offspring from conception to day 13 postpartum was not impaired at any dose level.
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 12 - 18, i.e. up to the day before necropsy (altogether for 55 or 64 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 for the intended subsequent histopathological examination.

Results:

MPI-ACA administered at 100, 300 or 1000 mg/kg bw/day did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Han: Wistar rats. The development of the F1 offspring from conception to day 13 postpartum after repeated oral administration was not impaired at any dose level.

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