Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined repeated dose toxicity study with the reproduction / developmental toxicity screening test - Gonads observations
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 13, 2015 to December 09, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
males body weight at arrival but this deviation was not considered to have affected the integrity or the purpose of the study
GLP compliance:
yes
Limit test:
no
Justification for study design:
This part will only focus on reproductive parameters. The systemic toxicity is assessed and described in the "repeated dose toxicity: oral" section.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there is ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
- 8 to 9 weeks old, weighing 225 to 235 g for males and 207 to 223 g for females, from Charles River Italia S.p.A.
- acclimatisation period of 33 days (main groups), 41 days (recovery groups), and ca. 70 days (positive control group).
- temperature and relative humidity at 22°C ± 2°C and 55% ± 15%, respectively.
- approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
- Drinking water was supplied ad libitum to each cage via water bottles. 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, except before blood samples withdrawal.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
softened
Details on exposure:
- The test substance was administered orally, by gavage to animals at 5 mL/kg bw suspended in softened water. The oral route was selected as it is a possible route of exposure of the test substance in man. Dose levels of 62.5, 250 and 500/1000 mg/kg bw/day were selected by the Sponsor based on information from previous studies. Control animals received the vehicle alone at the same dose volume. The positive control group (Group 7) received Mitomycin-C at a concentration of 2 mg/kg bw by intraperitoneal injection at the dose volume of 10 mL/kg body weight.
(The doses were administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.)

- Test substance
1. Main groups
The required amount was suspended in the vehicle (softened water, by reverse osmosis). The formulations were prepared daily at concentrations of 12.5, 50 and 200 mg/mL starting from Day 1 to Day 8 of the study. Starting from Day 9 of the study, the formulations were prepared daily at concentrations of 12.5, 50 and 200 mg/mL for males and 12.5, 50 and 100 mg/mL for females. Concentrations were calculated and expressed in terms of test substance as supplied.
2. Recovery groups
The required amount of was suspended in the vehicle (softenedwater, by reverse osmosis). Starting from Day 1 of the study, the formulations were prepared daily at concentrations of 12.5, 50 and 200 mg/mL for males and 12.5, 50 and 100 mg/mL for females. Concentrations were calculated and expressed in terms of test substance as supplied.
- Positive control substance
The required amount of positive control item was dissolved in the vehicle (sterile water for injection). The formulation was prepared on the day of dosing at a concentration of 0.2 mg/mL. Determination of the stability and concentration of solutions of the positive control substance were not undertaken.

- Each main group comprised 10 male and 10 female rats (Groups 1 to 4: 0, 62.5, 250, and 1000 mg/kg bw/day).
- Two groups (control and high dose levels) included 5 animals per sex to be sacrificed after 2 weeks of recovery (Groups 5 and 6).
- For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male and 5 female rats (Group7).
NB. In agreement with the Sponsor, the high dose level (1000 mg/kg bw/day) was reduced to 500 mg/kg bw/day in females of the main group, due to the toxicity observed during the first week of treatment (in terms of clinical signs, reduced body weight and food consumption). The dose level of 1000 mg/kg bw/day was administered to the animals (males and females) of the main groups starting from Day 1 to Day 8 of the study. Starting from Day 9 of the study for the main groups and from Day 1 for the recovery groups, the dose levels used were: 0, 62.5, 250 and 1000 mg/kg bw/day for males and 0, 62.5, 250 and 500 mg/kg bw/day for females.
Details on mating procedure:
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory (RTC Study No. A0773). The proposed formulation procedure for the test substance was checked in the range from 10 to 200 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period, to confirm that the method was suitable. Stability after 28 hours (10 mg/mL) and 26 hours (200 mg/mL) at room temperature and at +5°C for 8 days (range from 10 to 200 mg/mL) was also verified. In the present study, samples of the formulations prepared during the study were also analysed to check the concentration and homogeneity (two occasions during the study). Results of all analyses were within the acceptability limits for the first occasion. The results of the analysis in the second occasion were within range for Groups 1, 2 and 3 and below the acceptance range for Group 4. The analysis of a new preparation for this group gave acceptable results. Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A0773) using spectrophotometric analysis. The software used for this activity was SkanIt® version 2.4.2.55 (ThermoScientific).
Duration of treatment / exposure:
- Main groups (Groups 1 to 4):
Males animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Males were treated for a total of 37 or 38 days. Females animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post-coitum and post-partum periods until Day 3 post-partum (for at least 40 days). (During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post-coitum and on Day 1 post-partum. Thereafter individual dose volumes remained constant.)
- Recovery groups (Groups 5 and 6) animals were dosed once a day, 7 days a week, for 4 consecutive weeks. (No treatment was given during the recovery period.)
- Positive control group (Group7) animals received a single dose, approximately 24 h before sacrifice.
Frequency of treatment:
once a day, 7 days a week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
1000 to 500 mg/kg bw/day for females of the main groups; due to high toxicity
No. of animals per sex per dose:
10
(In addition, the micronucleus test was included (5 male and 5 female rats of the main groups) in order to assess the ability of the testsubstance to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes. The vehicle was
softened water, by reverse osmosis.)
Control animals:
yes, concurrent vehicle
Positive control:
Yes: mitomycin-C (i.p. injection)
Parental animals: Observations and examinations:
- Mortality: throughout the study, all animals were checked early in each working day in the morning and in the afternoon.
- Clinical signs: once before commencement of treatment and at least once daily during the study. Observations were performed at the same time interval each day (the interval was selected taking into consideration the presence of post-dose reactions (15-30 minutes after treatment).
- Clinical observations (Functional Observation Battery Tests)
Once before commencement of treatment and at least once a week from the start of treatment until termination. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
- Grip strength and sensory reactivity to stimuli: once during the study, towards the end of treatment.
- Motor activity assessment: once during the study, towards the end of treatment.
- Body weight:
Main groups. Males: weekly from allocation to termination and females: weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
Recovery groups. On the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Positive control group. On the day of allocation, on the day of dosing and just prior to necropsy.
- Food consumption: weekly.
- Clinical pathology investigations: samples of blood (for haematology and coagulation assessments, and clinical chemistry) and urine samples.
Oestrous cyclicity (parental animals):
- Vaginal smears: taken daily in the morning starting two weeks before pairing throughout the mating period until a positive identification of copulation was made. Determination of: 1. Anomalies of the oestrous cycle; 2. Pre-coital interval (i.e.,the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
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 also performed.
Litter observations:
Pups identification, weight and observation: as soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.
Postmortem examinations (parental animals):
- All females were examined for the following: 1. external and internal abnormalities; 2. number of visible implantation sites; 3. number of corpora lutea.
- The males were killed after the mating and all females, after a total of 37 or 38 days of dosing. Animals were killed after 2 weeks of recovery.The clinical history of adult animals was studied and a detailed post mortem examination was conducted (external and internal abnormalities, examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
- The ratios of organ weight to body weight were calculated for each animal and histopathological examinations were realised for the following organs::
Adrenal glands
Bone marrow (from femur)
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Clitoral gland
Caecum
Colon
Duodenum
Epididymides
Eye (+optin nerve)
Harderian glands
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Nasal cavity
Oesophagus
Ovaries with oviduct
Parathyroid grlands
Pituitary gland
Penis
Prostate gland
Rectum
Sciatic nerve
Seminal vesicles with coagulating glands
Spinal column
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach (forestomach and glandular)
Testes
Thymus (where present)
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina
Postmortem examinations (offspring):
All pups found dead in the cage were examined for external and internal abnormalities. All live pups at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
- Group mean values were calculated for all parameters. Data from non-pregnant females or total resorption were excluded from group mean calculations.
- Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified ttest, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) 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 significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
Copulation and fertility index, copulatory intervals, pre-implantation loss, and pre-natal loss.
Offspring viability indices:
Pup loss at birth, and post-natal loss on Day 4 past partum.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Main groups oestrous cycle and reproductive parameters (pre-coital intervals and copulatory indices) were similar in treated and control groups.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- Spermatogenic cycle:
A detailed qualitative examination of the testes was performed in five randomly selected control and high dose group males. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. The PAS-H stained sections were used to identify the spermatogenic stages. Testicular 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.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- Fertility index was decreased in treated males and females receiving the high dose level (1000/500 mg/kg bw/day).
- Implantation, pre-implantation loss data, pre-natal loss data and gestation length of females:
Gestation periods were similar between treated females and controls. All pregnant dams gave birth on Day 22 post coitum (mean value). A decrease in number of corpora lutea and implantation sites (-16% and -21%) was observed in treated groups (250 and 500 mg/kg bw/day) when compared to the control. Pre-implantation loss (percentage) was higher in one female receiving 500 mg/kg bw/day. In addition, a reduction in total litter size at birth (-23%) and consequently an increase in pre-natal loss (percentage) were also noted at this dose level (+29%) compared to control value. Moreover the total litter size was slightly reduced in females receiving 250 mg/kg bw/day. All these changes were not statistically significant.
(Three females receiving 1000 mg/kg bw/day for the first 8 days of treatment and 500 mg/kg bw/day for the rest of the study were found not pregnant at necropsy and one showed unilateral total resorption. In addition, one additional female receiving 62.5 mg/kg bw/day was found not pregnant. The number of females with live pups on Day 4 post partum was: 10 in the control, 9 in the low dose (62.5mg/kg bw/day), 10 in the mid-dose (250 mg/kg bw/day) and 6 in the high dose (500 mg/kg bw/day) groups.)
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Cold to touch, apparently no food intake (milk), small and pale appearance were the main clinical signs noted in control and treated pups. In addition, found dead or missing pups were observed both in control and treated groups, with the same incidence. One pup at the high dose level (500 mg/kg bw/day) showed bruise on head.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At birth and on Day 4 post partum, a decrease in live litter size and on Days 1 and 4 post partum, were observed in females receiving 250 (or more) mg/kg bw/day, when compared to control group without reaching statistical significance
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A decrease in litter weight (with significance at statistical analysis on Day 1 and 4 post partum) and mean foetal weight (with significance at statistical analysis on Day 4 post partum) were observed in females receiving 500 mg/kg bw/day, when compared to control group. A decrease in live litter size at birth and on Day 4 post partum and a decrease in litter weight on Days 1 and 4 post partum were also noted at 250 mg/kg bw/day.
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:
no effects observed
Description (incidence and severity):
Sex ratios at birth and on Day 4 post partum did not show differences between groups, when calculated as the percentage of males.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum:
No milk in the stomach autolysed and abdominal/thoracic organs were generally observed in a few pups died during the lactation period. No findings were noted in pups sacrificed on Day 4 post partum.
Histopathological findings:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Key result
Dose descriptor:
NOEL
Remarks:
teratogenic effects
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Conclusions:
Under the study conditions, the rat NOAEL for reproductive and developmental toxicity was considered to be 250 mg/kg bw/day for males and females.
Executive summary:

A study was conducted to determine the toxicity to reproduction of the test substance according to OECD Guideline 422, in compliance with GLP. Rats were administered the test substance orally by gavage at dose levels of 62.5, 250, 500, 1000 and 1000/500 mg/kg bw/day. Males animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Males were treated for a total of 37 or 38 days. Female animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post-coitum and post-partum periods until Day 3 post-partum (for at least 40 d). Recovery groups (0 or 1000, 1000/500 mg/kg bw/day test substance) animals were dosed once a day, 7 days a week, for 4 consecutive weeks. No treatment was given during the recovery period. Parental animals as well as litter and pups were extensively studied. During the in vivo phase of the study, the principal clinical sign recorded was staining of the body surface, particularly evident in males and females receiving the dose levels > 250 mg/kg body weight/day. This finding was also confirmed by brown staining or red faeces noted inside the cage, likely due to the compound eliminated by the animals. No findings of toxicological relevance were recorded at daily clinical observations including neurotoxicity assessment. Motor activity and sensory reaction to stimuli did not show relevant differences between control and treated groups. Lower body weights, body weight gains and food consumption were recorded in males and in females during the study at the dose levels > 250 mg/kg body weight/day, when compared to the control group. The principal alterations in haematological parameters were slight macrocytic anaemia, associated with moderate reticulocytosis noted in almost all animals receiving the high dose levels 1000/500 mg/kg body weight/day. Most of the males and one female receiving 250 mg/kg body weight/day showed also reticulocytosis. At clinical chemistry evaluation, some changes were noted at the end of the dosing period in animals receiving the dose levels > 250 mg/kg body weight/day: increase of cholesterol, protein, albumin and calcium and decrease of glucose levels in males and increase of cholesterol in females. Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day) and the copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage). Fertility index was decreased in treated males and females receiving the high dose level. A decrease in number of corpora lutea and implantation sites and an increase in pre-implantation loss (mean percentage) were observed in females dosed at 500 mg/kg body weight/day, when compared to the control. A reduction in total litter size at birth and consequently an increase in pre-natal loss (percentage) were also noted. Corpora lutea, implantation sites and a total litter size were reduced at 250 mg/kg body weight/day. These changes were however not statistically significant in both dose-groups. All pregnant females had a comparable length of gestation period and gave birth on Day 22 post-coitum (mean value). One high-dose female had unilateral total resorption. At birth and on Day 4 post partum, a decrease in live litter size and a statistically significant decrease in litter weight and mean foetal weight on Days 1 and 4 post-partum were observed in females receiving 500 mg/kg body weight/day, when compared to control group. A slight, however not statistically significant lower live litter size at birth and on Day 4 post-partum, and litter weight on Days 1 and 4 post-partum were also noted at 250 mg/kg body weight/day. Sex ratios at birth and on Day 4 post-partum did not show differences between groups, when calculated as the percentage of males. Similar clinical signs were recorded in control and treated pups during the lactation period. Necropsy findings in pups sacrificed at termination were comparable between groups. Deceased pups did not show relevant findings both in control and treated groups. At microscopic observation of parental animals, treatment-related changes in the liver (hepatocytic hypertrophy, associated with increased cytoplasmic eosinophilia), spleen (diffuse congestion, extramedullary haematopoiesis and yellow-brown pigment deposition) and kidneys (yellow-brown pigment granules, multifocally, observed in the cytoplasm of cortical tubular epithelial cells) were recorded in animals of both sexes and in the thyroids of only males receiving 1000/500 mg/kg body weight/day. At 250 mg/kg body weight/day, changes were noted in liver and thyroid in males and in the spleen in animals of both sexes. The findings observed in the liver of the high dose males (1000 mg/kg body weight/day) showed increased severity levels in comparison to mid dose males (250 mg/kg body weight/day) and occasionally they also showed hepatocytic degenerative changes (fatty change, hepatocytic necrosis, inflammatory reaction), often associated with follicular hypertrophy in the thyroid. Considering the pathological picture of the high dose males, the treatment-related changes observed in the liver could be considered adverse, while in males dosed at 250 mg/kg body weight/day, as well as in females dosed at 1000/500 mg/kg body weight/day, being not associated with any degenerative change, they were considered an adaptive change rather than an adverse effect. Follicular hypertrophy in the thyroids observed in most treated males, often associated with hepatocytic hypertrophy, was considered related to the liver drug-metabolizing enzyme induction.

In conclusion, under the study conditions, the rat NOAEL for reproductive and developmental toxicity was considered to be 250 mg/kg bw/day for males and females; as no malformations were observed in the offsprings, the NOEL for teratogenic effects considered to be 1000/500 mg/kg bw/day (Sisti, 2015).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A study was conducted to determine the toxicity to reproduction of the structural analogue according to OECD Guideline 422, in compliance with GLP. Rats were administered the test substance orally by gavage at dose levels of 62.5, 250, 500, 1000 and 1000/500 mg/kg bw/day. Males animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Males were treated for a total of 37 or 38 days. Female animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post-coitum and post-partum periods until Day 3 post-partum (for at least 40 d). Recovery groups (0 or 1000, 1000/500 mg/kg bw/day test substance) animals were dosed once a day, 7 days a week, for 4 consecutive weeks. No treatment was given during the recovery period. Parental animals as well as litter and pups were extensively studied. During the in vivo phase of the study, the principal clinical sign recorded was staining of the body surface, particularly evident in males and females receiving the dose levels > 250 mg/kg body weight/day. This finding was also confirmed by brown staining or red faeces noted inside the cage, likely due to the compound eliminated by the animals. No findings of toxicological relevance were recorded at daily clinical observations including neurotoxicity assessment. Motor activity and sensory reaction to stimuli did not show relevant differences between control and treated groups. Lower body weights, body weight gains and food consumption were recorded in males and in females during the study at the dose levels > 250 mg/kg body weight/day, when compared to the control group. The main alterations in haematological parameters were slight macrocytic anaemia, associated with moderate reticulocytosis noted in almost all animals receiving the high dose levels 1000/500 mg/kg body weight/day. Most of the males and one female receiving 250 mg/kg body weight/day showed also reticulocytosis. At clinical chemistry evaluation, some changes were noted at the end of the dosing period in animals receiving the dose levels > 250 mg/kg body weight/day: increase of cholesterol, protein, albumin and calcium and decrease of glucose levels in males and increase of cholesterol in females. Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day) and the copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage). Fertility index was decreased in treated males and females receiving the high dose level. A decrease in number of corpora lutea and implantation sites and an increase in pre-implantation loss (mean percentage) were observed in females dosed at 500 mg/kg body weight/day, when compared to the control. A reduction in total litter size at birth and consequently an increase in pre-natal loss (percentage) were also noted. Corpora lutea, implantation sites and a total litter size were reduced at 250 mg/kg body weight/day. These changes were however not statistically significant in both dose-groups. All pregnant females had a comparable length of gestation period and gave birth on Day 22 post coitum (mean value). One high-dose female had unilateral total resorption. At birth and on Day 4 post partum, a decrease in live litter size and a statistically significant decrease in litter weight and mean foetal weight on Days 1 and 4 post partum were observed in females receiving 500 mg/kg body weight/day, when compared to control group. A slight, however not statistically significant lower live litter size at birth and on Day 4 post partum, and litter weight on Days 1 and 4 post partum were also noted at 250 mg/kg body weight/day. Sex ratios at birth and on Day 4 post partum did not show differences between groups, when calculated as the percentage of males. Similar clinical signs were recorded in control and treated pups during the lactation period. Necropsy findings in pups sacrificed at termination were comparable between groups. Deceased pups did not show relevant findings both in control and treated groups.

At microscopic observation of parental animals, treatment-related changes in the liver (hepatocytic hypertrophy, associated with increased cytoplasmic eosinophilia), spleen (diffuse congestion, extramedullary haematopoiesis and yellow-brown pigment deposition) and kidneys (yellow-brown pigment granules, multifocally, observed in the cytoplasm of cortical tubular epithelial cells) were recorded in animals of both sexes and in the thyroids of only males receiving 1000/500 mg/kg body weight/day. At 250 mg/kg body weight/day, changes were noted in liver and thyroid in males and in the spleen in animals of both sexes. The findings observed in the liver of the high dose males (1000 mg/kg body weight/day) showed increased severity levels in comparison to mid dose males (250 mg/kg body weight/day) and occasionally they also showed hepatocytic degenerative changes (fatty change, hepatocytic necrosis, inflammatory reaction), often associated with follicular hypertrophy in the thyroid. Considering the pathological picture of the high dose males, the treatment-related changes observed in the liver could be considered adverse, while in males dosed at 250 mg/kg body weight/day, as well as in females dosed at 1000/500 mg/kg body weight/day, being not associated with any degenerative change, they were considered an adaptive change rather than an adverse effect. Follicular hypertrophy in the thyroids observed in most treated males, often associated with hepatocytic hypertrophy, was considered related to the liver drug-metabolizing enzyme induction.

In conclusion, under the study conditions, the rat NOAEL for reproductive and developmental toxicity was considered to be 250 mg/kg bw/day for males and females; as no malformations were observed in the offspring, the NOEL for teratogenic effects considered to be 1000/500 mg/kg bw/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed

Justification for classification or non-classification

Based on the results of the combined repeated dose and reproductive-developmental toxicity study in rats, no classification for reproductive toxicity is warranted for the test substance according to CLP (EC 1272/2008) criteria.

Additional information