Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD 421 study, no effects on reproduction performance have been noted up to 1000 mg/kg body weight.

The no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males based on the adverse changes in the thyroid glands.

The NOAEL for developmental toxicity in the offspring was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males due to retained areolae/nipples.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15.11.2016-9.11.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test (Jul 2000)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Females nulliparous and non-pregnant: yes
- Age at study initiation: male: 14-16 weeks, females: 12 weeks
- Average weight at study initiation: Males: 348.7 g - 415.7 g; Females: 186.6 g - 224.0 g
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III. Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, ad libitum
- Water: supplied from water bottles, ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% Sodium carboxymethyl cellulose suspension in drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations in 0.5% Sodium carboxymethyl cellulose suspension in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification. The preparations were kept at room temperature. For the test substance preparation, the specific amount of the test substance was weighed, topped up with 0.5% Sodium carboxymethyl cellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 dass
- Proof of pregnancy: vaginal sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in 0.5% Sodium carboxymethyl cellulose suspension in drinking water over a period of 7 days at room temperature were carried out prior to the start of the study. Samples of the test substance preparations were sent to the analytical laboratory twice during the study period for verification of the concentration (reserve samples collected at the beginning of the administration period and further samples collected during gestation). The samples which were taken for the concentration control analysis were also used to verify the homogeneity of the samples of the low- and high-concentration. From these concentrations three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running. Of each sample, one additional reserve sample was retained. Details of the sampling schedule were recorded with the raw data.
Duration of treatment / exposure:
The treatment lasted up to one day prior to sacrifice. The male and female animals were sacrificed 30 and 58 days, respectively, after the beginning of the administration, and examined.
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: yes
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume; therefore, these values are not reported in the Summary but in the Individual Tables.

FOOD CONSUMPTION
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14, and 14-20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OTHER:
Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. Blood samples were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Oestrous cyclicity (parental animals):
For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period. Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental
rats for a minimum of 2 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Litter observations:
STANDARDISATION OF LITTERS
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. All pups were examined externally and eviscerated, and their organs were assessed macroscopically.

PARAMETERS EXAMINED
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.
A check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups which died accidentally or had to be sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices was calculated according to the following formulas
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 13 after birth.
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. In the summary tables pup body weights and pup body weight gains are listed for males, females and males + females. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Anogenital distance (AGD) was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Blood samples the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Postmortem examinations (parental animals):
GROSS NECROPSY
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
Anesthetized animals, Epididymides, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands, Uterus (with cervix),
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

The following organs or tissues of all parental animals were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal glands, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964), Vagina

The testes and epididymides were histotechnical processed and examination by light microscopy in all control and high dose animals and in low and mid dose mating pairs suspected of reduced fertility. Ovaries, Liver and Thyroid glands were examined in all animals of all dose groups.
Postmortem examinations (offspring):
GROSS NECROPSY
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane by decapitation. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

HISTOPATHOLOGY / ORGAN WEIGTHS
On PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing (see chapter “Histopathology”).
Statistics:
- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means: Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index.
- Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups.
- Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development.
- Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians: Implantation sites, pups delivered, pups liveborn, live pups day x, viability index, survival index.
- Comparison of the dose group with the control group was performed using the WILCOXON test (twosided) for the hypothesis of equal medians: % live male day x, %live female day x
- Non-parametric oneway analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the equal medians: Number of cycles and cycle length, Blood parameters, Weight parameters
Reproductive indices:
Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index, Live birth index, Postimplantation loss,
Offspring viability indices:
Viability index, Survival index
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior which may be attributed to the test substance were detected in any male or female F0 generation parental animals at dose levels of 100 and 300 mg/kg bw/d during premating, gestation, lactation and post-mating periods. During lactation period, one high-dose female (No. 138 - 1000 mg/kg bw/d) showed salivation after treatment (within the 2-hour examination interval, PND 17-21). During the 5-hour examination interval (i.e. >2h<5h after treatment), no clinical signs or changes of general behavior were detected in this female. Therefore, this was assessed as spontaneous and not treatment related. No further clinical signs or findings were detected in any male or female F0 generation parental animals at dose levels of 1000 mg/kg bw/d during the different study periods. One sperm-positive mid-dose female (No. 123 - 300 mg/kg bw/d) did not deliver F1 pups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups (0, 100, 300 or 1000 mg/kg bw/d).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weights and mean body weight changes of all test substance treated F0 parental male and female animals (test groups 1-3) during the entire study period were comparable to the concurrent control values. The statistically significantly decreased mean body weight of the low-dose females (100 mg/kg bw/d) during GD 7 and 14 (up to 5% below control), as well as the significantly decreased mean
body weight change values of the low- and mid-dose females (100 and 300 mg/kg bw/d) during GD 0-7 (approx. 32% and 19% below control) were considered as spontaneous in nature and not treatment-related as no dose-response relationship occurred.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of the test substance-treated F0 males (test groups 1-3) was generally comparable to the respective control group during the entire treatment period. Food consumption of the F0 females in test group 2 and 3 (300 or 1000 mg/kg bw/d) was generally comparable to the respective control group during the entire treatment period. Food consumption of the F0 females in test group 1 (100 mg/kg bw/d) was statistically significantly decreased during gestation days 7-20 (up to 9% below control) and, as a consequence, during gestation days 0-20 (approx. 8% below control). During the other study periods (premating and lactation periods) the food consumption of the low-dose females was comparable to the concurrent control group. Thus, this sporadic event during gestation was considered as incidental and not treatment-related, as there was no dose-response relationship visible.
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, treatment-related
Description (incidence and severity):
After four-weeks of administration in F0 males thyroid stimulating hormone (TSH) levels were significantly increased and T4 levels were decreased although being not statistically significant. The TSH mean was above and that of T4 below the historical control range (males, TSH 5.07- 8.01 μg/L, T4 44.87-88.29 nmol/L). The corresponding hormone values in F0 females were not changed.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the liver and thyroid glands of males and females with incidences and grading according to the table below.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of test groups 0-3. The mean estrous cycle duration was statistically significantly decreased in test group 2: 4.00 / 4.47 / 3.78* / 3.97 days (p ≤ 0.05) in test groups 0-3, respectively. Although the mean value of test group 2 animals was outside the historical control data (cycles length: range of 3.99 – 4.46 days), there was no dose-response-relationship and, therefore, the finding was not related to treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100.0% in test groups 0-3. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One mid-dose male (No. 23 - 300 mg/kg bw/d) did not generate pregnancy. Thus, the male fertility index ranged between 90.0% and 100.0% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rat did not show relevant gross lesions.

The female mating index calculated after the mating period for F1 litter was 100.0% in all test groups. The mean duration until sperm was detected (GD 0) varied between 1.5 and 2.1 days without any relation to dosing. All female rats delivered pups or had implants in utero with the following exception:
• Mid-dose female No. 123 (mated with male No. 23) did not become pregnant.
The fertility index varied between 100.0% in test groups 0, 1 and 3 and 90.0% in test group 2. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The non-pregnant female had no relevant gross lesions. The mean duration of gestation was similar in all test groups (i.e. between 21.9 and 22.4 days). The gestation index was 100.0% in all test groups. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (11.6 / 12.0 / 12.7 and 13.1 implants/dam in test groups 0-3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (10.7 / 11.8 / 12.3 and 12.7 pups/dam in test groups 0-3, respectively). On average, test group 2 and 3 dams delivered 2 pups/litter more compared to control dams. The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 95.3% / 100.0% / 100.0% and 100.0% in test groups 0-3. Moreover, the number of stillborn pups was not significantly different between the test groups. Thus, the test substance did not adversely affect reproduction and delivery of the F0 generation parental females.
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The mean number of delivered F1 pups per dam and the rates of liveborn F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
- Pup viability: The viability index indicating pup mortality during early lactation (PND 0-4) varied between 99.1% (test group 2), 99.3% (test groups 1 and 3) and 100.0% (control) without relation to dosing. The survival index indicating pup mortality during PND 4-13 varied between 100.0% / 97.5% / 100.0% / 100.0% in test groups 0-3, respectively.
- Pup mortality: The pup mortality shown in Figure 4.2.2.1.3.1 is based on total number of stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights of the test substance-treated F1 male and female pups (and both sexes combined) were below the concurrent control group during the entire lactation period, attaining statistical significance on different time points:
On PND 1, the mean body weights of the high-dose male pups (1000 mg/kg bw/d) were statistically significantly decreased (about 9% below control).
On PND 4, a statistically significant decrease in body weight was also observable in male pups and both sexes combined in test groups 1-3 (100, 300 or 1000 mg/kg bw/d - up to 13% below control), in female pups in test groups 2 and 3 (about 12% below control).
All values were within the historical control range (PND 1: male: 5.4 – 7.4 g; PND 4: male: 8.1 – 11.8 g, female: 8.0 – 11.2 g, both sexes: 8.1 – 11.4 g).
The following statistically significant decrease of pup body weights was observed on the further days:
• On PND 7, the mean body weight of the female and male pups and both sexes combined in test groups 1 and 3 (up to 10% below control)
• On PND 13, the mean body weights of the male F1 pups in test group 3, in female pups and both sexes combined in test groups 1 and 3 (up to 8% below control, respectively).
The mean body weight change of all test substance-treated male and female F1 pups (and both sexes combined) was statistically significantly decreased on PND 1-4 (about 18% below control, both sexes combined), furthermore, in the low- and high-dose female pups and in both sexes combined, on PND 1-13 (about 8% below control).

In general, control dams had a smaller litter size compared to the test groups 1-3 dams. Consequently, control pups exhibited higher body weights based on the low pup numbers compared to the test group pups. The mean numbers of F1 pups delivered were 10.7 / 11.8 / 12.3 and 12.7 pups/dam in test groups 0-3, respectively. On average, test groups 2 and 3 dams delivered 2 pups/litter more compared to control dams. For the test groups, dams had to take care of a higher number of pups and, therefore, the individual weight of the pups were lower compared to control.
Overall, the decrease in pup body weight in the respective test groups was due to differences in litter size. In cases where historical control data are available, all values were within the range of historical control data. Furthermore, the decrease in pup body weights on PND 7 and 13 was not dose-dependent since body weights of test group 2 pups were not altered. Therefore, the decrease in pup body weights of test groups 1-3 were not considered as treatment-related and adverse.
One male runt was seen in the control, one female runt was seen in test group 3, one male and one female runt was seen in test groups 1 and 2. The findings were not assessed as treatment-related since they were observed only in single pups or were not related to dose.
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:
effects observed, non-treatment-related
Description (incidence and severity):
In F1 PND13 female pups of test group 13 (1000 mg/kg bw/d), T4 values were significantly decreased with no significant changes of TSH. However, the T4 mean was within the historical control range (females, PND13 T4 41.14-75.73 nmol/L). No hormone changes were observed in PND13 males.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (100, 300 and 1000 mg/kg bw/d). The statistically significantly decreased anogenital distance of male pups in test group 1 (100 mg/kg bw/d) is considered as spontaneous in nature due to the lack of dose-response relationship.
Nipple retention in male pups:
effects observed, treatment-related
Description (incidence and severity):
The percentage of male pups having nipple / areolae was statistically significantly increased in test group 3 (1000 mg/kg bw/d) when examined on PND 13 (44.7% / 67.3% / 78.7% / 90.0%*
[p ≤ 0.05] in test groups 0-3). The mean value was outside the historical control range (males with areolae/nipples: range of 0.0 – 81.7 %). The mean number of nipple / areolae in the respective male pups was also statistically significantly increased in test group 3 (1.2 / 1.8 / 2.1 / 2.5* [p ≤ 0.05] in test group 0-3). The mean value was on the border of the historical control data (nipple number, range of 0.0 – 2.5). Due to the dose-related increase of male pups having nipples/areolae and the statistically significant increase in both parameters (mean number of nipple / areolae and percentage of affected males), a relation to treatment cannot be excluded. Therefore, the findings concerning nipple development in males of test group 3 were assumed to be treatment-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few F1 pups showed spontaneous findings at gross necropsy, such as partly cannibalized, small testis and dilated renal pelvis. These findings occurred without any relation to dosing and/or some of them can be found in the historical control data at comparable incidences. None of the findings was considered to be related to the treatment.
Description (incidence and severity):
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups. The statistically significantly increased number of male pups in test group 3 and decreased number of female pups on PND 0 was considered to be spontaneous in nature. The mean values of the males and females were within the range of the historical control data (live males: 36.6 – 60.5 %; live females: 39.5 – 63.4 %). The concurrent control group showed, however, a rather low number of male pups which may have led to this statistical significance without having any biological relevance. Therefore, the finding was not assessed as treatment-related.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: development, retained areolae/nipples
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effect
Critical effects observed:
not specified
Reproductive effects observed:
no

Absolute and relative organ weights

Absolute organ weights

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly increased (statistically significant changes printed in bold):

Absolute weights Males Females
Test group (mg/kg bw/day) 1
(100)
2
(300)
3
(1000)
1
(100)
2
(300)
3
(1000)
Liver 107% 104% 119%** 94% 103% 117%**
Ovaries       96% 114%* 117%**
Heart       88%** 99% 98%

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights

When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased (statistically significant changes printed in bold):

Relative weights Males Females
Test group (mg/kg bw/day) 1
(100)
2
(300)
3
(1000)
1
(100)
2
(300)
3
(1000)
Liver 105%* 104% 121%** 100% 107% 119%**
Ovaries       101% 117%* 120%**
Thyroid gland       96% 108% 123%*

The significant absolute and relative liver weight increase in males and females of test group 3 was regarded as treatment-related and correlated with centrilobular hepatocellular hypertrophy.

The significant absolute weight increase of the ovaries in females of test groups 2 and 3 (118.3 and 121.5 mg, respectively) was within the historical control range (101.2 – 125.0 mg). However, the relative weight increase in test group 3 (0.054 %) was minimally above the historical control range (0.042 – 0.052%), whereas in test group 2 the relative weight increase was weak significant and was within the historical range (0.052 %). Since no histopathological correlate was found in the ovaries to explain these weight deviations, the significant weight increase in test groups 2 and 3 was assumed to be incidental.

The significant relative weight increase of the thyroid glands (0.01%) in females of test group 3 was above the historical control range (0.007 – 0.009%) and correlated with histopathological findings.

The significant relative weight increase of the liver in test group 1 males and the significant absolute weight decrease of the heart in test group 1 females occurred both incidentally and were not considered treatment-related. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Histopathology

Treatment-related findings were observed in the liver and thyroid glands of males and females with incidences and grading according to the table below:

LIVER

Male animals Female animals
Test group (mg/kg bw/day) 0
(0)
1
(100)
2
(300)
3
(1000)
0
(0)
1
(100)
2
(300)
3
(1000)
No. of animals 10 10 10 10 10 10 10 10
Liver                
Hypertrophy, centrilobular 0 0 2 8 0 0 1 10
·    Grade 1     2 7     1 7
·    Grade 2       1       3

The centrilobular hepatocellular hypertrophy in males and females of test group 2 and 3 was considered treatment-related but not adverse.

THYROID GLAND

  Male animals Female animals
Test group (mg/kg bw/day) 0
(0)
1
(100)
2
(300)
3
(1000)
0
(0)
1
(100)
2
(300)
3
(1000)
No. of animals 10 10 10 10 10 10 10 10
Thyroid gland                
Hypertrophy / hyperplasia, follicular 2 3 8 8 0 2 1 4
·    Grade 1 2 3 4 3   2 1 4
·    Grade 2     4 5        
Altered colloid 3 4 6 5 2 3 2 4
·    Grade 1 3 3 2 2 1 2 2 3
·    Grade 2   1 4 3 1 1   1

The hypertrophy/hyperplasia of follicular cells and altered colloid in males of test group 2 and 3 was considered treatment-related although no dose-dependent relationship in incidence and/or grading occurred between both groups. In females of test group 3, a minimal increase in the incidence of the hypertrophy/hyperplasia of follicular cells and the altered colloid was assumed to be treatment-related.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Thyroid Hormones

Males:

  Test Group 0/ M 0 mg/kg bw/d Test Group 1/ M 100 mg/kg bw/d Test Group 2/ M 300 mg/kg bw/d Test Group 3/ M 1000 mg/kg bw/d
T4 [nmol/L] day 30 Mean 50.10 k 43.80 45.03 40.92
S.d. 8.22 5.78 7.54 5.36
N 10 10 10 10
Median 47.21 44.11 46.96 39.91
Deviation Vs Control   -12.58 -10.12 -18.31
TSH [µg/L] day 30 Mean 8.87 v 8.69 13.79 12.44 *
S.d. 2.50 2.19 7.43 3.36
N 10 10 10 10
Median 8.91 8.57 11.41 12.30
Deviation Vs Control   -1.96 55.46 40.26

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKAL-WALLIS

Females:

  Test Group 0/ F 0 mg/kg bw/d Test Group 1/ F 100 mg/kg bw/d Test Group 2/ F 300 mg/kg bw/d Test Group 3/ F 1000 mg/kg bw/d
T4 [nmol/L] day 49 Mean 50.81 k 48.11 51.23 49.22
S.d. 7.79 5.18 6.41 9.63
N 10 10 9 10
Median 48.83 48.7 49.44 47.60
Deviation Vs Control   -5.32 0.83 -3.13
TSH [µg/L] day 49 Mean 6.92 k 7.39 6.96 7.75
S.d. 2.44 2.84 3.01 1.70
N 10 10 9 10
Median 6.46 7.04 6.46 7.63
Deviation Vs Control   6.78 0.61 12.07

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKAL-WALLIS

Male Pups

  G 10 / M G 11 / M G 12 / M G 13 / M
T4 [nmol/L] day 13 Mean 70.67 k 62.87 66.72 60.25
S.d. 10.79 8.04 7.22 9.27
N 10 10 9 10
Median 70.11 63.64 66.96 59.42
Deviation Vs Control   -11.04 -5.59 -14.75
TSH [µg/L] day 13 Mean 3.80 k 3.90 4.36 4.45
S.d. 0.86 0.87 1.07 1.06
N 10 10 9 10
Median 3.76 3.98 4.02 4.28
Deviation Vs Control   2.63 14.56 16.96

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKAL-WALLIS

Female Pups

  G 10 / F G 11 / F G 12 / F G 13 / F
T4 [nmol/L] day 13 Mean 69.61 v 63.27 69.28 57.89 *
S.d. 11.07 11.03 5.81 8.07
N 10 10 9 10
Median 71.72 61.38 69.68 57.14
Deviation Vs Control   -9.11 -0.47 -16.84
TSH [µg/L] day 13 Mean 3.88 k 4.24 4.28 4.29
S.d. 0.79 1.18 1.69 0.65
N 10 10 9 10
Median 3.77 3.93 3.74 4.48
Deviation Vs Control   9.25 10.40 10.62

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics v=KRUSKAL-WALLIS-WILCOX; k=KRUSKAL-WALLIS

Conclusions:
In conclusion, under the conditions of the present Reproductive/Developmental Toxicity Screening Test the oral administration of the test item to male and female Wistar rats revealed signs of systemic toxicity such as adverse changes in thyroid hormones and corresponding hypertrophy/hyperplasia of follicular cells and altered colloid in the thyroid glands of adult males at 1000 mg/kg bw/d. Females showed no signs of systemic toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males based on the adverse changes in the thyroid glands. The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the offspring was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males due to retained areolae/nipples.
Executive summary:

The test article was given daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose suspension in drinking water [0.5% CMC]). The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, one day post-mating in males, and the entire gestation period as well as approximately 20 days of the lactation period in females. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. Food consumption of the F0 parental animals was determined once weekly during premating. In dams food consumption was determined for gestation days (GD) 0-7, 7-14, 14-20 and lactation days (PND) 1-4, 4-7, 7-10 and 10-13. Body weights of F0 parental animals were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two-week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. Anogenital distance measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. At necropsy on PND 4 and PND 13, the pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. On PND 13 thyroid glands and parathyroid glands were fixed for possible further processing. All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Blood samples for clinical pathological investigations were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 during necropsy of pups. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The following test substance-related adverse effects/findings were noted: In the high dose group, increased TSH values and decreased T4 values were reported in adult males, correlating with hypertrophy/hyperplasia in follicular cells of the thyroid in 8 out of 10 males (minimal to slight) and altered colloid in 5 out of 10 males (minimal to slight). No test substance-related adverse findings in females. In male pups of the high dose group the percentage of male pups having nipple / areolae was statistically significantly increased (90.0 %, control: 44.7 %) and the mean number of nipples in males was increased (2.5, control: 1.2) on PND 13. No adverse findigs were reported for the mid and low dose groups.

In conclusion, under the conditions of the present Reproductive/Developmental Toxicity Screening Test the oral administration of the test item to male and female Wistar rats revealed signs of systemic toxicity such as adverse changes in thyroid hormones and corresponding hypertrophy/hyperplasia of follicular cells and altered colloid in the thyroid glands of adult males at 1000 mg/kg bw/d. Females showed no signs of systemic toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males based on the adverse changes in the thyroid glands. The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the offspring was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males due to retained areolae/nipples.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 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

The test item was administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at doses of 100, 300 and 1000 mg/kg bw/d. The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as 13 days of lactation in females.

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was also demonstrated over a period of 7 days at room temperature. No signs of general systemic toxicity (clinical findings, food consumption, body weight) were observed in male and female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d). Fertility, reproductive performance and delivery were not impaired by test substance up to a dose of 1000 mg/kg bw/d, as was demonstrated by unchanged fertility, gestation and live birth indices of pups in all test groups. The viability and survival indices as indicator for pup mortality were also not significantly altered. Concerning nipple development in male pups, percentage of affected males and mean number of male pups having nipple / areolae was statistically significantly increased in test group 3 (1000 mg/kg bw/d) on PND 13. The sex ratio on day of birth showed no indication for an increase in female pups. The number of male pups was even increased but was within the historical control range. However, since both parameters of nipple development were doserelated, statistically significantly altered, a relation to treatment cannot be excluded and, therefore, this was considered to be treatment related and adverse. Regarding clinical pathology, a hypothyroid effect in adult males of test group 3 (1000 mg/kg bw/d) cannot be excluded because of changes in thyroid hormone values (increased TSH and decreased T4 values). No effect on thyroid hormone levels was observed in adult females as well as in F1 PND13 pups. Regarding pathology, significant absolute and relative liver weight increases in male (absolute: +19 %; relative: +21 %) and female animals (absolute +17 %; relative: +19 %) of test group 3 (1000 mg/kg bw/d) were consistent with centrilobular hepatocellular hypertrophy in both male (minimal to slight) and female (minimal to slight) animals. These findings were regarded as treatment-related but not adverse. In the thyroid gland of males of test group 3, hypertrophy/hyperplasia of follicular cells and altered colloid were clearly increased in incidence and grading, when compared to control group animals. Since these findings correlated with increased TSH and decreased T4 values, they were considered treatmentrelated and adverse. In females of test group 3, a statistically weak significant increase of the relative thyroid gland weight correlated with a minimal increase in the incidence of hypertrophy/hyperplasia of follicular cells and altered colloid. Since these findings occurred without hormonal changes, they were assumed to be treatment-related but not adverse. In test group 2 (300 mg/kg bw/d), the incidence of centrilobular hepatocellular hypertrophy in the liver was very low in both male (two out of 10) and female animals (one out of 10). This finding was regarded as possible treatment-related but not adverse. In the thyroid gland of males, hypertrophy/hyperplasia of follicular cells and altered colloid were increased and comparable to the changes seen in test group 3. However, since these changes were not associated with TSH and T4 alterations, they were regarded as possible treatment-related but not adverse. In test group 1 (100 mg/kg bw/d), no treatment-related findings were seen in the liver and thyroid gland of male and female animals. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Under the conditions of the present Reproductive/Developmental Toxicity Screening Test the oral administration of the test item to male and female Wistar rats revealed signs of systemic toxicity such as adverse changes in thyroid hormones and corresponding hypertrophy/hyperplasia of follicular cells and altered colloid in the thyroid glands of adult males at 1000 mg/kg bw/d. Females showed no signs of systemic toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males based on the adverse changes in the thyroid glands. The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the offspring was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males due to retained areolae/nipples.

Effects on developmental toxicity

Description of key information

A PNDT study is currently conducted following ECHA Decision Number: CCH-D-2114440074-60-01/F. Once the study is completed, a dossier update will be submitted.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

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

The available screening study is reliable. It is as suitable for classification purposes under Regulation 1272/2008 and as a result the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).