Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-18 to 2018-01-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-18 to 2018-01-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650:
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Batch identification: 0013479406
Species:
rat
Strain:
Wistar
Remarks:
strain Crl:WI(Han)
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 13-14 weeks
- Weight at study initiation: Mean weight per group: Male: 391.6 g - 394.7 g; Female: 213.9 g - 219.1 g
- Housing: Individually during the study period; During overnight matings, male and female mating partners were housed together; Pregnant animals and their litters were housed together until PND 13
The bedding and the enrichment is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals) by the producer.
- Diet: Kliba maintenance diet mouse/rat, ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
Food analyses
The food used in the study was assayed for chemical and for microbiological contaminants.
With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable.

Drinking water analyses
The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities.
On the basis of the analytical findings, the drinking water was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % suspension in drinking water with 5 mg/100 mL Tween 80
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a
calibrated beaker depending on the dose group, topped up with 0.5 % Carboxymethylcellulose suspension in drinking water with 5 mg/ 100mL Tween 80 and intensely mixed with a magnetic stirrer.
During administration, the preparations were kept homogeneous with a magnetic stirrer

VEHICLE
- Justification for use and choice of vehicle: The test substance is stable in the vehicle.
- Concentration in vehicle: 1 g/100 mL; 3 g/100 mL; 10 g/100 mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method: Quantitative LC/ESI-MS with evaluation by the external standard method.

Analytical verifications of the stability of the test substance in 0.5 % Carboxymethylcellulose suspension in drinking water (with 5 mg/ 100mL Tween 80) for a period of 7 days at room temperature were carried out before the study was initiated.
At the beginning (during premating) and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control
analysis.
The samples collected at the beginning of the administration period and the first set of samples during lactation were analyzed in the Analytical Laboratory at the Competence Center Analytics.
Due to unexpected high values in the measured analytical samples of the mid and low dose group at the Competence Center Analytics a more sensitive analytical method was used for the remaining samples collected during the study. The additional samples were measured at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology.

The stability of test substance in 0.5 % Carboxymethylcellulose suspension in drinking water + 5 mg/100mL Tween 80 was demonstrated for a period of 7 days at room temperature.
In summary it can be stated that the homogeneous distribution of the test substance in the vehicle was demonstrated and the measured values for the test substance were basically in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: By request of the sponsor
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.
- Cage side observations checked: Signs of morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In all animals once before the start of the administration period and thereafter at weekly intervals. (Parameters measured see below table no. 1)

BODY WEIGHT: Yes
- Time schedule for examinations: 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.

HAEMATOLOGY: Yes
- Time schedule for collection of blood and no. of animals: From all F0 parental male animals of all test groups shortly before sacrifice and from all F0 parental female animals of all test groups on PND 14
- How many animals: From all F0 parental male animals of all test groups, all F0 parental female animals of all test groups
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- Parameters checked in table no 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning
- Animals fasted: Yes
- How many animals: The first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Parameters checked in table no.3 were examined.

Thyroid Hormones
Blood samples from all dams at PND 14 and all males at termination.
Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 1. On study day 30; 2. On study day 56
- Dose groups that were examined: 1. Five male animals per group; 2. Five female animals (with litter) per group
- Battery of functions tested: followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests (see table no. 8 A)- C)).
Motor activity measurement : The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was determined once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
- Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals)
- Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 7-8, 14-15 and 20-21.
- Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 4-5, 7-8, 10-11 and 13-14.

FOOD CONSUMPTION: Yes
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 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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The male and female animals were sacrificed 34 and 58/62 days, respectively, after the beginning of the administration, and examined.

ORGAN WEIGHTS
See table no. 4 and 5

Organ/tissue fixation (all parental animals) see table no.6

HISTOPATHOLOGY: Yes (see table no.7)

Statistics:
Blood parameters (clinical pathology)
Statistical test: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians.

Weight parameters (pathology)
Statistical test: Non-parametric one-way 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 (two-sided) for the equal medians.

Water consumption, food consumption (parental animals), body weight and body weight change (parental animals)
Statistical test: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All high- and mid-dose male and female F0 animals showed discolored feces (orange) from premating day 1 (test group 3) and 3 (test group 2) till the end of the study.
All high-dose male and female F0 animals showed discolored urine (yellow) from premating day 10 till the end of the study.
Several male animals of test groups 3 showed salivation after treatment (grade: slight to severe) during several parts of the mating period and during the entire post-mating period.
During a part of the gestation period one female of test group 3 and during several parts of the lactation period several females of test group 3 showed salivation after treatment (grade: slight to moderate).
One mid-dose female (No.122) showed a swelling at the right forelimb from the end of premating till the end of the study because of an injury.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the low-dose male and female F0 animals during the entire study period.

Detailed clinical observations
All male and all female animals of all dose groups (100, 300 and 1000 mg/kg bw/d) did not show any abnormalities.
One mid-dose female (No.122) showed a swelling on the right forelimb because of an injury.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
One animal of dose group 1 (No. 117 - 300 mg/kg bw/d) was sacrificed premature on premating day 6 because it escaped from the cage during the first premating week [one week before start of the mating period] and paired with male animal No. 18.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights of all male and female parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study period.
This includes the statistically significantly decreased values during premating days 7 - 13 in test groups 1-3 and the statistically significantly increased values in the high-dose females during premating days 0 - 7.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of all male and female F0 animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption of all male and female F0 animals of all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control values throughout the entire study.
The supposedly decreased water consumption of all test substance-treated F0 females during one single timepoint at the end of the lactation period (PND 13 – 14) was assessed as incidental.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
In males of test group 3 (1000 mg/kg bw/d) absolute and relative lymphocyte counts were increased (absolute counts not statistically significant). However, both parameter values were within historical control range (males, absolute lymphocytes 3.16-5.96 Giga/L; relative lymphocytes 70.0-82.0 %). In males of test group 2 (300 mg/kg bw/d) relative monocyte counts were significantly lower compared to controls, but the change was not dose-dependent.
Therefore, the mentioned changes were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.

In females of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d), calcium levels were significantly higher compared to controls. The calcium medians and means of test groups 1 and 3 were within, those of test group 2 above, and mean and median of the controls below the historical control range (females, calcium 2.56-2.66 mmol/L). Calcium alterations in the test groups were not dose-dependently changed. Alanine aminotransferase (ALT) activities in males of test group 2 (300 mg/kg bw/d) were significant lower compared to controls, but the change was also not dose-dependent. Therefore, the mentioned alterations among clinical chemistry parameters were regarded as incidental and not treatment-related.

Thyroid hormones
In parental males of test group 2 (300 mg/kg bw/d) significant higher T4 levels compared to controls were observed.
However, this change was not dose-dependent and therefore it was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.

Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups.

Motor activity measurement (MA)
No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Minor changes in thymus, adrenal gland, kidney and thyroid gland weight are considered to be of no significant toxicologic relevance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
An orange yellow discoloration of the content of the gastrointestinal tract (particularly of the stomach, jejunum, cecum or colon) was observed in 2/19 animals of the 100 mg/kg/d, 5/20 animals of the 300 mg/kg/d and 16/20 animals of the 1000 mg/kg/d treated group. The highest incidence of this finding was found in the glandular stomach.
The discoloration of the gastrointestinal content, supposed to be related to the chemical properties of the test item could not be related to any histopathologic change of the mucosa or underlying structures.
Except for the discolored gastrointestinal content, all gross findings in this study are considered spontaneous, incidental or related to technical procedures and consistent with the usual pattern of findings in animals of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The discoloration of the gastrointestinal content, supposed to be related to the chemical properties of the test item could not be related to any histopathologic change of the mucosa or underlying structures.
Except for the discolored gastrointestinal content, all histopathologic findings in this study are considered spontaneous, incidental or related to technical procedures and consistent with the usual pattern of findings in animals of this strain and age.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance related adverse effects were observed.
Key result
Critical effects observed:
no

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650:
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch identification: 0013479406

Test animals

Species:
rat
Strain:
Wistar
Remarks:
strain Crl:WI(Han)
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P)13-14 weeks
- Weight at study initiation: Mean weight per group: Male: 391.6 g - 394.7 g; Female: 213.9 g - 219.1 g
- Housing: Individually during the study period; During overnight matings, male and female mating partners were housed together; Pregnant animals and their litters were housed together until PND 13
- Diet: Kliba maintenance diet mouse/rat, ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
Food analyses
The food used in the study was assayed for chemical and for microbiological contaminants.
With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable.

Drinking water analyses
The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities.
On the basis of the analytical findings, the drinking water was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % suspension in drinking water with 5 mg/100 mL Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a
calibrated beaker depending on the dose group, topped up with 0.5 % Carboxymethylcellulose suspension in drinking water with 5 mg/ 100mL Tween 80 and intensely mixed with a magnetic stirrer.
During administration, the preparations were kept homogeneous with a magnetic stirrer

VEHICLE
- Justification for use and choice of vehicle: The test substance is stable in the vehicle.
- Concentration in vehicle: 1 g/100 mL; 3 g/100 mL; 10 g/100 mL
- Amount of vehicle: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Pregnant animals and their litters were housed together until PND 13
- Any other deviations from standard protocol:
Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
Exception: One animal of dose group 1 (No. 117 - 300 mg/kg bw/d) escaped from the cage during the first premating week [one week before start of the mating period] and paired with male animal No. 18. Therefore the data from female 117 could no longer be used and the animal was sacrificed premature on premating day 6.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method: Quantitative LC/ESI-MS with evaluation by the external standard method.

Analytical verifications of the stability of the test substance in 0.5 % Carboxymethylcellulose suspension in drinking water (with 5 mg/ 100mL Tween 80) for a period of 7 days at room temperature were carried out before the study was initiated.
At the beginning (during premating) and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control
analysis.
The samples collected at the beginning of the administration period and the first set of samples during lactation were analyzed in the Analytical Laboratory at the Competence Center Analytics.
Due to unexpected high values in the measured analytical samples of the mid and low dose group at the Competence Center Analytics a more sensitive analytical method was used for the remaining samples collected during the study. The additional samples were measured at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology.

The stability of test substance in 0.5 % Carboxymethylcellulose suspension in drinking water + 5 mg/100mL Tween 80 was demonstrated for a period of 7 days at room temperature.
In summary it can be stated that the homogeneous distribution of the test substance in the vehicle was demonstrated and the measured values for the test substance were basically in the expected range of the target concentrations (90 - 110 %), demonstrating the correctness of the preparations.
Duration of treatment / exposure:
2 weeks: male 33 days, female 57/61 day
Frequency of treatment:
once daily
Details on study schedule:
No pups were selected for mating as a OECD 422 was conducted.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: By request of the sponsor
Positive control:
not conducted

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.
- Cage side observations checked: Signs of morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In all animals once before the start of the administration period and thereafter at weekly intervals. (Parameters measured see below table no. 1)

BODY WEIGHT: Yes
- Time schedule for examinations: 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.
- 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) and on PND 4, 7, 10 and 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood and no. of animals: From all F0 parental male animals of all test groups shortly before sacrifice and from all F0 parental female animals of all test groups on PND 14
- How many animals: From all F0 parental male animals of all test groups, all F0 parental female animals of all test groups
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- Parameters checked in table no 2 were examined.

CLINICAL CHEMISTRY
- Time schedule for collection of blood: In the morning
- Animals fasted: Yes
- How many animals: The first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Parameters checked in table no.3 were examined.

Thyroid Hormones
Blood samples from all dams at PND 14 and all males at termination.
Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 1. On study day 30; 2. On study day 56
- Dose groups that were examined: 1. Five male animals per group; 2. Five female animals (with litter) per group
- Battery of functions tested: followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests (see table no. 8 A)- C)).
Motor activity measurement : The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was determined once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
- Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals)
- Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 7-8, 14-15 and 20-21.
- Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 4-5, 7-8, 10-11 and 13-14.

FOOD CONSUMPTION: Yes
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 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.
Oestrous cyclicity (parental animals):
For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization.
For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. 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.
Sperm parameters (parental animals):
Parameters examined in all/ male parental generations: Testis weight, epididymis weight
Special attention was given on the stages of spermatogenesis in the testes (Histopathology)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); Surplus pups or 2 preferably female pups per litter were sacrificed

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: - number and sex of pups, stillbirths, live births,
- macroscopically evident changes,
- postnatal mortality (twice daily on workdays (once in the morning and once in the afternoon, only in the morning on Saturdays, Sundays or public holidays),
- Anogenital distance (AGD) was determined in all live male and female pups on PND 1 (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).
- Presence of nipples/areolae in male pups: All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.

Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 10 and 13.



Postmortem examinations (parental animals):
SACRIFICE
The male and female animals were sacrificed 34 and 58/62 days, respectively, after the beginning of the administration, and examined.

GROSS NECROPSY
- Gross necropsy consisted of external examinations special attention being given to the reproductive organs.

ORGAN WEIGHTS
See table no. 4 and 5

Organ/tissue fixation (all parental animals) see table no.6

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues/ organs indicated in table no. 5-7 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
As result of standardization surplus pups were sacrificed at 4 days of age.
The surplus pups or 2 preferably female pups per litter, respectively, were sacrificed under isoflurane anesthesia by decapitation

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.
At necropsy on PND 13, 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.

GROSS NECROPSY
- Gross necropsy consisted of external examinations and internal including macroscopic assessment of organs.

Statistics:
Please refer to "any other information on materials and methods incl. tables"
Reproductive indices:
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility*/ number of males placed with females) x 100
* defined by a female with implants in utero

The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

Female mating index (%) = (number of females mated*/ number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of females pregnant*/ number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth/ number of females pregnant*) x 100
* defined as the number of females with implants in utero
Offspring viability indices:
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:

Live birth index (%) = (number of liveborn pups at birth/ total number of pups born) x 100

The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:

Postimplantation loss (%) = (number of implantations – number of pups delivered/ number of implantations) x 100

In general, 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 by the methods, which are described in detail in section “Necropsy observations”.
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 after birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices was calculated according to the following formulas:

Viability index (%) = (number of live pups on day 4* after birth/ number of live pups on the day of birth) x 100
* before standardization of litters (i.e. before culling)

Survival index (%) = (number of live pups on day 13 after birth/ number of live pups on day 4* after birth) x 100
* after standardization of litters (i.e. after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All high- and mid-dose male and female F0 animals showed discolored feces (orange) from premating day 1 (test group 3) and 3 (test group 2) till the end of the study.
All high-dose male and female F0 animals showed discolored urine (yellow) from premating day 10 till the end of the study.
Several male animals of test groups 3 showed salivation after treatment (grade: slight to severe) during several parts of the mating period and during the entire post-mating period.
During a part of the gestation period one female of test group 3 and during several parts of the lactation period several females of test group 3 showed salivation after treatment (grade: slight to moderate).
One mid-dose female (No.122) showed a swelling at the right forelimb from the end of premating till the end of the study because of an injury.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the low-dose male and female F0 animals during the entire study period.

Detailed clinical observations
All male and all female animals of all dose groups (100, 300 and 1000 mg/kg bw/d) did not show any abnormalities.
One mid-dose female (No.122) showed a swelling on the right forelimb because of an injury.
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.
One animal of dose group 1 (No. 117 - 300 mg/kg bw/d) was sacrificed premature on premating day 6 because it escaped from the cage during the first premating week [one week before start of the mating period] and paired with male animal No. 18.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights of all male and female parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study period.
This includes the statistically significantly decreased values during premating days 7 - 13 in test groups 1-3 and the statistically significantly increased values in the high-dose females during premating days 0 - 7.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of all male and female F0 animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption of all male and female F0 animals of all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control values throughout the entire study.
The supposedly decreased water consumption of all test substance-treated F0 females during one single timepoint at the end of the lactation period (PND 13 – 14) was assessed as incidental.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
In males of test group 3 (1000 mg/kg bw/d) absolute and relative lymphocyte counts were increased (absolute counts not statistically significant). However, both parameter values were within historical control range (males, absolute lymphocytes 3.16-5.96 Giga/L; relative lymphocytes 70.0-82.0 %). In males of test group 2 (300 mg/kg bw/d) relative monocyte counts were significantly lower compared to controls, but the change was not dose-dependent.
Therefore, the mentioned changes were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.

In females of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d), calcium levels were significantly higher compared to controls. The calcium medians and means of test groups 1 and 3 were within, those of test group 2 above, and mean and median of the controls below the historical control range (females, calcium 2.56-2.66 mmol/L). Calcium alterations in the test groups were not dose-dependently changed. Alanine aminotransferase (ALT) activities in males of test group 2 (300 mg/kg bw/d) were significant lower compared to controls, but the change was also not dose-dependent. Therefore, the mentioned alterations among clinical chemistry parameters were regarded as incidental and not treatment-related.

Thyroid hormones
In parental males of test group 2 (300 mg/kg bw/d) significant higher T4 levels compared to controls were observed.
However, this change was not dose-dependent and therefore it was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.

Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups.

Motor activity measurement (MA)
No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The discoloration of the gastrointestinal content, supposed to be related to the chemical properties of the test item could not be related to any histopathologic change of the mucosa or underlying structures.
Except for the discolored gastrointestinal content, all histopathologic findings in this study are considered spontaneous, incidental or related to technical procedures and consistent with the usual pattern of findings in animals of this strain and age.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
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 all test groups 0 - 3. The mean estrous cycle duration was similar: 4 days in test groups 0 - 3, respectively.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
One sperm negative low-dose female (100 mg/kg bw/d - No. 111) did not deliver F1 pups.
This was considered not be attributed to the treatment as in the mid and high dose group all couples had offspring and also because in none of the dose groups morphological findings in the reproductive organs of either sex were found which could be attributed to the test item. The spermatogenic staging profiles were normal for all males examined.
Reproductive performance:
no effects observed
Description (incidence and severity):
Male reproduction data
For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Copulation was not confirmed for low-dose male No. 11 paired with low-dose female No. 111. Thus, the male mating index was 88.9 % in the low-dose group and 100 % in the control, mid- and high-dose groups.

Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. The above mentioned low-dose male (300 mg/kg bw/d - No. 11) did not generate pregnancy. Thus, the male fertility index ranged between 88.9 % and 100 % without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 88.9 % in the lowdose group and 100 % in the control, mid- and high-dose groups.
The mean duration until sperm was detected (GD 0) varied between 2.2 and 3.6 days without any relation to dosing.
All female rats delivered pups with the following exception:
- Low-dose female No. 111 (mated with male No. 11) did not become pregnant.

The fertility index was 100 % in all test groups 0 - 3.
The non-pregnant low dose female No. 111 had inflammatory changes in the vaginal wall.
It cannot be ruled out that these inflammatory changes had influence on the mating behavior. But nevertheless it is considered as an incidental finding and not related to the test-item.

The mean duration of gestation values varied between 22.0 (control and test group 1), 22.1 (tests group 2) and 22.2 (test group 3).
The gestation index was 100 % in all test groups 0 - 3.

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 (13.3 / 14.4 / 13.2 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 (12.0 / 13.0 / 12.1 and 12.3 pups/dam in test groups 0 - 3, respectively)
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100 % / 98.1 % / 100 % and 96.7 % in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance related adverse effects were observed.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized 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 lactation (PND 0 - 4) varied between 100 %/ 98.3 % / 99.3 % and 100 % in test groups 0 - 3, respectively.
The pups surviving index indicating pup mortality during lactation (PND 4 - 13) varied was 100 % in test groups 0 - 3, respectively.

Pup mortality
The pup mortality shown based on total number of stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups:
Females: 6 (PND 0-4)
Males: 3 (PND 0)
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related influence on body weights and body weight change values of F1 pups were noted in test groups 1 - 3 (100, 300 and 1000 mg/kg bw/d).
One male runt was seen in the control and one female runt was seen in test group 2 and 3, respectively.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Thyroid hormones
In male and female pups at PND13 of test groups 11, 12 and 13 (100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 levels were observed.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Anogenital distance/anogenital index
Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (test groups 1 - 3 [100, 300 and 1000 mg/kg bw/d]).

Nipple/ areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Concerning pathology the daily repeated oral administration of the test item did not result in premature decedents, nor in relevant changes in terminal body and organ weights (minor changes in thymus, adrenal gland, kidney and thyroid gland weight are considered to be of no significant toxicologic relevance).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, such as discolored testis (left, red), dilated renal pelvis and partly cannibalized.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all
these findings were not considered to be associated to the test substance.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Sex ratio:
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance related adverse effects were observed.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Reproductive parameter indices

Test group (mg/kg bw/d)

Test Group 0/ F

0 mg/kg bw

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

P0 Males

 

 

 

 

Mating index

100

88.9

100

100

Fertility index

100

88.9

100

100

P0 Females

 

 

 

 

Mating index

100

88.9

100

100

Fertility index

100

100

100

100

Gestation index

100

100

100

100

F1 Pups

 

 

 

 

Viability index

100

98.3

99.3

100

Applicant's summary and conclusion