Registration Dossier

Administrative data

Description of key information

The NOAEL for general toxicity was determined to be 200 mg/kg bw/d (reference 7.5.1 -1).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2017 - May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation: males: 362 - 405 g (mean: 381.23 g, ± 20% = 304.98 - 457.47 g), females: 223 - 268 g (mean: 244.12 g, ± 20% = 195.30 - 292.95 g)
- Housing: Animals were housed in groups of 2 animals / sex / cage in type III polysulphone IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqua ad iniectabilia (sterile water), Manufacturer: AlleMan Phama
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item, as delivered, was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved. Contact with glass was prevented due to the characteristics of the test item (adherence to glass). The test item formulations were prepared freshly on each administration day before the administration procedure. Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: 702240
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Agroscience Services EcoChem GmbH. The test item was shown to be homogeneous and stable for 6 hours according to Eurofins Agroscience Services EcoChem GmbH Analytical Phase Code S17-06310-L2. Samples were collected during the study for the investigation of homogeneity (samples from the top, middle and bottom) and of substance concentration in study week 1 (pre-mating period) week 3 (first week of mating), week 5 (gestation) and in the last week of the study (gestation / lactation) from all groups. In parallel a sample of control item formulation was sampled in the respective week (22 samples).Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Agroscience Services EcoChem GmbH Analytical Phase Code S17-06310-L2 and until then stored under appropriate conditions based on available stability data. The B-samples will be retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
70 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
100 animals (40 males and 60 females) were included in the study. 60 females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were continued in the study. Remaining not selected 20 females were discarded without any observations or used for other appropriate study.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose range finding study; In the range finding study mortality was observed at the highest dose level (1000 mg/kg bw/d). Therefore the next lower dose (200 mg/kg bw) was selected as highest dose in the main study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day. Twice daily all animals were observed for morbidity and mortality.
- Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, and PND 13 along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION: Yes
- Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- The following parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc), prothrombin time (PT) and activated partial thromboplastin time (aPTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment prior to or as part of the sacrifice of the animals
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each group
- The following parameters were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na) and potassium (K).

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment prior to or as part of the sacrifice of the animals. Additionally, urine color/ appearance was recorded.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the week before the first treatment and during the last week of the treatment
- Dose groups that were examined: 5 randomly selected males and during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group.
- Battery of functions tested: sensory activity / grip strength / motor activity and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

THYROID HORMONES (T4):
- Time schedule for examinations: at termination
- How many animals: all adult males
Sacrifice and pathology:
GROSS NECROPSY: Yes
- All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY: Yes (see list below)
- Adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (axillary), lymph nodes (mandibular), lymph nodes (mesenteric), mammary gland area (male and female), oesophagus, optic nerves, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid/parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina
Other examinations:
ORGAN WEIGHTS: Yes (see list below)
- Testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), uterus with cervix, ovaries (paired weight), thyroid/parathyroid glands, thymus, liver, spleen, kidneys (paired weight), brain, adrenal (paired weight), heart and pituitary gland.
Statistics:
A statistical assessment of the results of body weight and food consumption was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Low incidences of slight clinical signs like alopecia in one male of the MD group, one male of the HD group, two female animals of the control group, and one female animal of the MD group were seen without dose dependency and are not considered test item-related.
Moving the bedding was observed transiently in few females (7/10) of the HD group and salivation was noted transiently in 1/10 males of the HD group. The clinical signs of moving the bedding and salivation were mainly seen at the end of the treatment period in males and during the lactation period in females. Both signs were observed in short timely relation to dose administration or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction of the test item. These slight signs were not considered as adverse systemic effects. Regurgitation was observed in a single animal on a single day (GD 13) and is not considered related to toxicity but to the oral gavaging procedure.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the treatment period in any of the test item-treated groups and the control group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The test item had no effect on body weight development of male and female animals in this study. Body weights developed normally. There were no statistically significant differences between dose groups and control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
During the entire study period, there was no considerable and statistically significant difference in food consumption of male and female animals between the dose groups and the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The test item had no effect on haematological parameters and coagulation parameters of male and female animals in this study. There were no statistically significant differences between dose groups and control group.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The test item had no effect on clinical biochemistry parameters and coagulation parameters of male and female animals in this study. There were no statistically significant differences between dose groups and control group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment all parameters of urinalysis of the test item-treated groups period were not considerably different compared to the corresponding control group and were within the normal range of variation.
Single high values of erythrocytes or protein values in LD or HD groups were considered incidental and without relation to the treatment with the test item.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared to the controls. There were no biologically relevant differences in body temperature between the groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute and relative spleen weight was non-significantly higher in male animals of the LD, the MD, and the HD group when compared to the control group (absolute LD deviation from control: 39 %, absolute MD deviation from control: 39 %, absolute HD deviation from control: 39%). This was independent of dose and not associated with any test item-related histopathological findings. Thus, it is not considered toxicologically relevant.
A tendency towards higher absolute and relative ovary weight in female animals of the LD group (deviation from control: 21 %) was noted. As reproductive organs undergo variable changes depending on the estrous cycle, the finding showed no dose-dependency, and was not associated with a test item-related histopathological finding this effect is not considered related to treatment with the test item.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Only incidental findings were noted at necropsy of the animals of the parental generation. A thickened wall of the left uterus horn of control female no. 42, a dilated uterus of MD female no. 66, and an enlarged spleen of HD female no. 72 are not considered to be test item-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross lesions or histological findings that could be attributed to treatment with the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Treatment with the test item had no effect on serum T4 levels of males of the parental generation or on 13 day old pups. No considerable differences were found between dose groups and control group of this study.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No treatment-related adverse effects were observed.
Key result
Critical effects observed:
no

Dose Formulation Analysis

Concentration analysis of chromium in formulation samples was determined at three concentrations, 145 mg/L, 507 mg/L and 1448 mg/L in study weeks 1, 3, 5 and in the last week of the study. The mean recoveries observed for the LD dose group was between 99 % and 108 % of the nominal value, between 99 % and 104 % for the MD dose group and between 98 % and 105 % of the nominal value for HD dose group.

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15 %.

Homogeneity of formulation samples was determined at three Chromium concentrations, 145 mg/L, 507 mg/L and 1448 mg/L, in study weeks 1, 3, 5 and the last week of the study.

Conclusions:
The NOAEL for repeated dose toxicity was determined to be 200 mg/kg bw/d.
Executive summary:

The aim of this study according to OECD guideline 422 was to assess the possible effects of the test item on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study. The following doses were evaluated: 0 (control), 20, 70 and 200 mg/kg body weight/day. The test item formulation was prepared freshly on each day of administration. The test item was prepared with aqua ad injectionem (sterile water) and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 12 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 10 mL/kg body weight. During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five randomly selected males and females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment from all animals and in the last week of treatment in five randomly selected males and females of each group. After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on post-natalday 4 or 13and those found dead, were carefully examined for gross external abnormalities. A full histopathological evaluation of the preserved tissues (adrenal glands, testes, epididymides, ovaries, oviducts, uterus with cervix, vagina, prostate and seminal vesicle with coagulating gland) was performed on high dose and control animals, in non-pregnant female animals and male mating partners of the low and medium dose. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. All gross lesions macroscopically identified were examined microscopically in all animals.

No mortality occurred during the treatment period with the test item in any of the test item-treated groups or the control group. There were no clinical signs of systemic toxicity in this study. Moving the bedding and increased salivation were noted in few animals of the high dose group and were assumed to be a sign of discomfort or a local reaction of the test item but not toxicologically relevant. No toxicologically relevant effects were observed in any of the parameters of the functional observation battery when comparing test item-treated groups to their corresponding controls. Treatment with the test item had no effect on body weight development or food consumption in this study. Parameters of haematology, coagulation, clinical biochemistry and urinalysis were not affected by treatment with the test item. The test item had no effect on organ weights determined at terminal necropsy. There were no gross lesions or histological findings that could be attributed to treatment with the test item. The incidence of a higher number of abnormal cycles was observed in all groups without showing a dose dependency and thus was not considered toxicologically relevant. The test item had no test item-related effect on the precoital interval and the duration of gestation throughout the dose groups. Pre-natal and post-natal parameters including the number of corpora lutea, the number of implantations and the number of live pups on PND 0, 4, and 13 were not affected by treatment with the test item. Reproductive indices such as the copulation index, fertility index, delivery index, and viability index showed no test item-related effects. Slight differences were within the range of historical control data. Treatment with the test item had no toxicologically relevant effects on the number of pups, still births, runts, the number of live pups, male and female pups and the sex ratio on both, PND 4 and PND 13, when test item-treated groups with the control group. Mean pup weight was not affected and total litter weight, male litter weight, and female litter weight were comparable between groups which received test item and the control group. Anogenital distance was slightly but statistically significantly smaller in the HD pups when compared to controls. As this is an incidental finding which is not associated with other abnormalities, it is not considered to be toxicologically relevant. Treatment with the test item had no toxicologically relevant effect on serum T4 levels of parental males or the 13 day old pups. Slight statistically significant higher thyroid/parathyroid weight of male pups compared to controls was not considered toxicologically relevant. Isolated external pup findings observed in this study were not considered test item-related.

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test in male and female Wistar rats with dose levels of 20, 70, and 200 mg/kg bw/d the following conclusions can be made: No signs of systemic toxicity was found at dose levels up to the maximum feasible dose of 200 mg/kg bw/d. Therefore the NOAEL for general toxicity is considered to be 200 mg/kg bw/d. A slightly lower anogenital distance found at the HD level when compared to controls is considered an isolated and non-adverse finding. In the absence of other correlating contributing factors for effect on developmental and reproductive toxicity in terms of effect on parental histopathology, pup weight, pup survival, reproductive and developmental indices, hormone analysis, estrus cyclicity, the NOAEL for this reproduction/developmental toxicity screening study may be considered to be 200 mg/kg bw/d.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The aim of this study according to OECD guideline 422 was to assess the possible effects of the test item on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study. The following doses were evaluated: 0 (control), 20, 70 and 200 mg/kg body weight/day. The test item formulation was prepared freshly on each day of administration. The test item was prepared with aqua ad injectionem (sterile water) and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 12 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 10 mL/kg body weight. During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five randomly selected males and females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment from all animals and in the last week of treatment in five randomly selected males and females of each group. After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on post-natalday 4 or 13and those found dead, were carefully examined for gross external abnormalities. A full histopathological evaluation of the preserved tissues (adrenal glands, testes, epididymides, ovaries, oviducts, uterus with cervix, vagina, prostate and seminal vesicle with coagulating gland) was performed on high dose and control animals, in non-pregnant female animals and male mating partners of the low and medium dose. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. All gross lesions macroscopically identified were examined microscopically in all animals.

No mortality occurred during the treatment period with the test item in any of the test item-treated groups or the control group. There were no clinical signs of systemic toxicity in this study. Moving the bedding and increased salivation were noted in few animals of the high dose group and were assumed to be a sign of discomfort or a local reaction of the test item but not toxicologically relevant. No toxicologically relevant effects were observed in any of the parameters of the functional observation battery when comparing test item-treated groups to their corresponding controls. Treatment with the test item had no effect on body weight development or food consumption in this study. Parameters of haematology, coagulation, clinical biochemistry and urinalysis were not affected by treatment with the test item. The test item had no effect on organ weights determined at terminal necropsy. There were no gross lesions or histological findings that could be attributed to treatment with the test item. The incidence of a higher number of abnormal cycles was observed in all groups without showing a dose dependency and thus was not considered toxicologically relevant. The test item had no test item-related effect on the precoital interval and the duration of gestation throughout the dose groups. Pre-natal and post-natal parameters including the number of corpora lutea, the number of implantations and the number of live pups on PND 0, 4, and 13 were not affected by treatment with the test item. Reproductive indices such as the copulation index, fertility index, delivery index, and viability index showed no test item-related effects. Slight differences were within the range of historical control data. Treatment with the test item had no toxicologically relevant effects on the number of pups, still births, runts, the number of live pups, male and female pups and the sex ratio on both, PND 4 and PND 13, when test item-treated groups with the control group. Mean pup weight was not affected and total litter weight, male litter weight, and female litter weight were comparable between groups which received test item and the control group. Anogenital distance was slightly but statistically significantly smaller in the HD pups when compared to controls. As this is an incidental finding which is not associated with other abnormalities, it is not considered to be toxicologically relevant. Treatment with the test item had no toxicologically relevant effect on serum T4 levels of parental males or the 13 day old pups. Slight statistically significant higher thyroid/parathyroid weight of male pups compared to controls was not considered toxicologically relevant. Isolated external pup findings observed in this study were not considered test item-related.

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test in male and female Wistar rats with dose levels of 20, 70, and 200 mg/kg bw/d the following conclusions can be made: No signs of systemic toxicity was found at dose levels up to the maximum feasible dose of 200 mg/kg bw/d. Therefore the NOAEL for general toxicity is considered to be 200 mg/kg bw/d. A slightly lower anogenital distance found at the HD level when compared to controls is considered an isolated and non-adverse finding. In the absence of other correlating contributing factors for effect on developmental and reproductive toxicity in terms of effect on parental histopathology, pup weight, pup survival, reproductive and developmental indices, hormone analysis, estrus cyclicity, the NOAEL for this reproduction/developmental toxicity screening study may be considered to be 200 mg/kg bw/d.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The NOAEL for general systemic toxicity was determined to be 200 mg/kg bw/d. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.