Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 22, 2017 to February 1, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation:
♂ 200 to 225 g
♀ 175 to 200 g
- Fasting period before study:
- Housing: From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 4 2.5×26.6×18.5 cm with a stainless steel mesh lid and floor ( Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×1 8.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week.
- Diet: A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: Drinking water was supplied ad libitum to each cage via water bottles
- Acclimation period: approximately 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55 % ± 15 %
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): and the rooms were lit by artificial light for 12 hours each day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Samples of the formulations prepared on Week 1 and last week were analysed to check the homogeneity and concentration.

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg body weight
Details on mating procedure:
Mating was monogamous (one male to one female).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A 28 hour stability at room temperature and an 8 day stability at +5 °C ± 3 °C were verifi ed in the range from 10 to 200 mg /ml.
Duration of treatment / exposure:
Males: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter for a total of 46/47 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing a nd thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifi ce( for at least 51 days). Dose volumes were adjusted once per week for each animal
according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Recovery groups: (Groups 5 and 6)
Animals were dosed once a day, 7 days a week, for 4 consecutive weeks. Dose volumes were adjust ed once per week for each animal according to the last recorded body weight. No treatment was given during the recovery period.
Frequency of treatment:
Animals were dosed once a day, 7 days a week
Details on study schedule:
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter for a total of 46/47 days.
The female was paired with the same male until positive identifi cation occurred or 14 days had elapsed.

Parturition and gestation length (Main groups)
A parturition check was performed from Day 20 to Day 25 post coitum. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth. The day of birth (when parturition was complete) is defined as Day 0 post partum.

Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Main group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Main group
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Main group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Main group
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Recovery group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Recovery group
No. of animals per sex per dose:
Each main group comprised 10 male and 10 female rats. Two groups (control and high dose levels) included 6 animals per sex to be sacrificed after 4 weeks of recovery.
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
MORTALITY: Yes
Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the fi nal check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHT: Yes
Main groups:
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identifi cation of mating and on
Days 0, 7, 14 and 20 post coitum. Dams and pups were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy. Recovery groups:
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION:
Main groups:
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum. Recovery groups:
The weight of food consumed by each cage of rats was recorded at weekly intervals following Day 1 of dosing.

Blood collection from parental animals (Main and Recovery groups): Yes
Males
Blood samples were collected under isofl uorane anaesthesia from the retro-orbital sinus. The order of collection was equalised between groups.
Females
As part of the sacrifi cial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava. The order of collection was equalised between groups.
Blood collection was performed only for animals at termination.

Euthanasia
Parental animals were killed by exsanguination under isofluorane anaesthesia.
Parental males (Main groups)
The males were killed after the mating of all females, after a total of 46/47 days of treatment.
Parental females (Main groups)
The females with live pups were killed on Day 14 post partum, after a total of 51 days of treatment.
Males and females (Recovery groups)
Animals were killed after 4 weeks of recovery.

Necropsy (All groups)
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifi ces). Changes were
noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
Females (Main groups)
All females were examined also for the following:
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of apparently non-pregnant females were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Histopathological examination (Main and Recovery groups)
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
In the first instance, the examination was restricted as detailed below:
- Tissues specifi ed in section 4.5.8 from all males and all females in the control and high dose groups killed at term.
- All abnormalities in all groups.
Oestrous cyclicity (parental animals):
Stock females
Oestrous cycle was monitored by vaginal smears in all stock females for 1 week before allocation in order to exclude from the study females with irregular cycle.
Females allocated to groups (Main groups)
Vaginal smears were taken in the morning from Day 1 of dosing up to positive identifi cation of mating.
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken from all females, before despatch to necropsy. No vaginal smears were taken from females sacrifi ced for humane reasons.
Sperm parameters (parental animals):
During the necropsy procedure, one cauda from one epididymis of each parental male completing the scheduled test period in the high dose and control group was taken for sperm count and evaluation of motility, morphology and viability. The animals of the mid and low dose groups were dosed until the evaluation of the control and high dose animals had been performed.
Sperm analysis of mid- and low dose animals completing the scheduled test period was not carried out, since no differences were seen between the control and high dose animals.
Litter observations:
Pups identifi cation, weight and observation (Main groups)
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4, 7 and 13 post partum.

Observation was performed once daily for all litters.
Pups dying during the lactation period were weighed before the despatch to necropsy. After culling, all pups were sacrifi ced with the dams on Day 14 post partum.

Blood collection from pups on Days 4 and 14 post partum: Yes,
On Day 4 post partum, as a part of the necropsy procedure, blood samples (approximately 0.2 ml) were taken from 2 pups (1 male and 1 female, where possible). On Day 14 post partum, blood samples (approximately 0.5 mL) were taken from 2 pups (1 male and 1 female, where possible). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture). The order of collection was equalised between groups.
Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots for analysis. The aliquots obtained were stored at -80°C until analysis.

Bioanalysis - Thyroid hormone determination (T3, T4 and TSH)
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using Luminex Magpix system and the MILLIPLEX MAP Rat Thyroid Magnetic Bead Panel kit (Merk Millipore, cat. no. RTHYMAG-30K). In the first instance the determination was restricted as detailed below:
– Samples from all parental males from all main groups
– Samples from pups on Day 14 post partum
The results of these analyses are presented as individual data, mean and standard deviations.
Since no treatment related effects were seen in the determination performed in parental males and in pups on Day 14 post partum, samples obtained from females and from pups on Day 4 post partum were not analysed and will be destroyed after the fi nalisation of the report. When the serum levels for Total triidothyronine (total T3) were BLOQ (below the limit of quantifi cation - 3.75 ng /ml), the values were not included in the mean calculation.

Euthanasia
Pups were euthanised by intraperitoneal injection of Thiopenthal.

Necropsy
All pups were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrifi ced on Day 14 post partum were killed and examined for external abnormalities and sex confi rmation by gonadal inspection. All pups with abnormalities were retained in 10% neutral buffered formalin.
Postmortem examinations (parental animals):
A parturition check was performed from Day 20 to Day 25 post coitum. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth. The day of birth (when parturition was complete) is defi ned as Day0 post partum.
Postmortem examinations (offspring):
All pups were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrifi ced on Day 14 post partum were killed and examined for external abnormalities and sex confi rmation by gonadal inspection. All pups with abnormalities were retained in 10% neutral buffered formalin.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the signifi cance of the differences amongst group means was assessed by Dunnett’s test or a modifi ed t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test, if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical signifi cance wasp <0.05.
Reproductive indices:
During the necropsy procedure one cauda from one epididymis of each parental male completing the scheduled test period in the high dose and the control group will be taken for sperm count and evaluation of motility, morphology and viability.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No signs of toxicity were observed throughout the study, both in main and recovery groups. The stain ing of the tail observed in all animals receiving 500 and 1000 mg/kg/day and in animals after 4 weeks of recovery, was related to the colour of the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred throughout the study, both in main and recovery groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Main groups:
No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.
Recovery groups:
Means of body weight and body weight gain were comparable between controls and the treated groups both in males and females throughout the treatment phase. During the recovery period, no relevant differences in body weight and body weight gain were recorded in animals of both sexes, when compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Main and recovery groups
No effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid stimulating hormone was decreased in males of all treated groups. Compared with controls, changes were 39% to 46%, with no dose-relation. Due to the absence of related changes of the other thyroid hormones and/or histopathological fi ndings, these were considered to be of no toxicological relevance.
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:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in animals sacrificed at the end of the treatment period. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
The sporadic lesions reported in control and/or treated animals such as epithelial hyperplasia of non glandular region of the stomach in one high dose male (no. X0850080) or luminal dilatation of the uterus of one high dose female (no. X0850079), were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age. In particular, the M-Adenocarcinoma in the mammary glands of one mid-dose female (no. X0850053), which correlated to the subcutaneous mass observed during necropsy, was considered spontaneous.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cycle and copulatory index were similar between treated and control animals.
The statistically signifi cant increase of pre-coital interval detected in the high dose group was considered incidental since no dose-relation was noted.
Fertility index both for males and females was 100% in the control and mid-dose groups and 90% in the low and high dose groups. Two control males (nos. X0850038 and X0850080)
failed to induce pregnancy. These cases were considered incidental.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No relevant differences were observed at sperm analysis including sperm motility and concentration, and cauda weight between the control and the high dose group at the end
of treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
Copulatory index were similar between treated and control animals. The statistically signifi cant increase of pre-coital interval detected in the high dose group was considered incidental since no dose-relation was noted.
Fertility index both for males and females was 100% in the control and mid-dose groups and 90% in the low and high dose groups. Two control males (nos. X0850038 and X0850080)
failed to induce pregnancy. These cases were considered incidental.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no releated effects observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
Small appearance and pallor were the main clinical signs noted in control and treated pups. Found dead or missing pups were also observed both in control and treated groups. No
toxicological signifi cance was attributed to these fi ndings which are commonly observed in the litters during the lactation period.
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No relevant changes were observed on terminal body, absolute and relative organ weight of treated animals that completed the treatment or recovery period, when compared to
the controls. The sporadic statistically signifi cant changes observed in some organs (i.e. increased relative kidneys weight in high dose males (appr. 10% of relative mean weight) or
females (appr. 7% of relative mean weight)) sacrifi ced at the end of treatment or recovery, or in terminal body weight of the recovery phase were not considered toxicologically relevant.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no releated effects observed

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Remarks on result:
not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Remarks on result:
not measured/tested

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general and reproduction/developmental toxicity was considered to be 1000 mg/kg/day.
Executive summary:

The toxic effects on rats of both sexes after repeated oral dosing with test substance, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring were

investigated. A 4 week treatment-free period was allowed in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase.

The vehicle was softened water. All doses were administered at a constant volume of 5 ml/kg body weight.

The study design was as follows:

Main groups
Group  Treatment
(mg/kg/day)
Number of animals
1 0 10♂ + 10♀
2 150 10♂ + 10♀
3 500 10♂ + 10♀
4 1000 10♂ + 10♀
Recovery groups
Group  Treatment
(mg/kg/day)
Number of animals
5 0 6♂ + 6♀
6 1000 6♂ + 6♀

Main groups

Males of the main groups were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 46/47 days. Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days).

The following investigations were performed in both sexes of all groups: mortality check, clinical signs, body weight, food consumption, oestrous cycle, mating performance, litter data, sex ratios, macroscopic observations and organ weights. Sperm analysis was performed in all control and high dose males. Histopathological examination was performed in control and high dose groups, as well as on all abnormalities detected during post mortem observation. The identification of the stages of the spermatogenic cycle was also performed in five randomly selected males of the control and high dose groups. Thyroid hormone determination was performed in all males. Clinical signs were performed in all pups. Thyroid weight and thyroid hormone determination of 1/pup/sex/litter (where possible) at Day 14 post partum were evaluated. All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination.

Sex was determined by internal gonads inspection. All live pups sacrificed on Day 14 post partum were examined for external abnormalities and sex confirmation

by gonadal inspection. The anogenital distance in all pups and the presence of nipples/areolae in male pups were also verified.

Recovery groups

Recovery animals were treated for 4 consecutive weeks and killed after 4 weeks of recovery period. The following parameters were evaluated in these animals: mortality check, clinical signs, body weight, food consumption, macroscopic observations and organ weights. Mortality and fate of females

No mortality occurred throughout the study. Mating was not detected in one female of the mid-dose group. However, this female gave birth and was sacrificed with the litter on Day 14 post partum.

One female in the low dose group and one female in the high dose group were found not pregnant at necropsy. In addition, one female in the high dose group had unilateral implantation.

The number of females with live pups on Day 14 post partum was: 10 in the control, 9 in the low dose, 10 in the mid-dose and 9 in the high dose group.

Clinical signs

Main and recovery groups. No signs of toxicity were observed throughout the study, both sexes, both for main and recovery groups.

Body weight and body weight gain

Main and recovery groups. No differences of toxicological relevance were recorded in body weight and body weight gain, both for main and recovery groups respect to the control groups.

Food consumption

Main and recovery groups. No effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.

Thyroid hormone determination

No treatment-related differences were noted in hormones levels in parental males and in pups at Day 14 post partum, when compared to control group animals.

Oestrous cycle, reproductive parameters, pairing combination and mating performance Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show any differences that could be related to treatment.

Implantation, pre-birth loss data and gestation length of females

Corpora lutea, implantations, litter size and pre-natal loss (percentage) were similar in control and treated groups. Gestation periods were similar between treated and control females. All pregnant dams gave birth on Day 22 post coitum (mean value).

Litter data and sex ratio of pups

Litter data at birth on Days 1, 4 and 13 post partum did not show differences of toxicological relevance between groups. Sex ratios at birth, on Days 4 and 14 post partum did not show relevant differences between groups, when calculated as the percentage of males.

Clinical signs of pups

Pre-weaning clinical signs, pups found dead and missing pups were comparable between treated and control groups.

Anogenital distance

No relevant differences were seen between the control and treated pups in anogenital distance.

Necropsy findings in pups

Necropsy findings in deceased pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.

Pup thyroid weight

No significant differences were noted in mean thyroid weight of pups in control and treated groups.

Terminal body weight and organ weights

Main and recovery groups

No relevant changes were observed on terminal body weight, absolute and relative organ weight of treated animals that completed the treatment or recovery period, when compared with controls.

Sperm analysis

No relevant differences were observed at sperm analysis including sperm motility and concentration, and cauda weight between the control and the high dose group at the end of treatment.

Macroscopic observations

Main and recovery groups. No remarkable differences were noted at post mortem examination in treated animals sacrificed at the end of treatment and recovery periods, when compared with controls.

Microscopic observations

No treatment-related changes were noted in animals sacrificed at the end of the treatment period. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Conclusion

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general and reproduction/developmental toxicity was considered to be 1000 mg/kg/day.