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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:
05 June 2012 to 07 August 2012
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
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
05 June 2012 to 07 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
22 March 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA 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:
Lot/batch No.: 110007P040
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 wks
- Weight at study initiation: on average: Males: 335 g; Females: 197 g
- Fasting period before study: no
- Housing: single (except during mating and during lactation) in Markrolon type M III cages
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal ad lib.
- Water: ad lib.
- Acclimation period: app. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The desired amount of test substance was weighed, and corn oil was added up to the correct volume. To prepare a homogenous suspension, the mixture was stirred with a magnetic stirrer also during administration. The test substance preparations were produced at least once a week and were stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle: The test substance is poorly soluble in water, but forms a homogenous suspension in corn oil, which is also non-toxic to rats.
- Concentration in vehicle: 1.25; 3.75; 12.5 g/100 mL
- Amount of vehicle: 4 mL/ kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90.0-110.0% of the nominal concentrations. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that teh test substance was distributed homogeneously in corn oil.
Duration of treatment / exposure:
Males: 35 days
Females: 56 days
Frequency of treatment:
daily (except to animals being in labor)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 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: The selection of doses was based on the results of a test study in female Wistar rats (BASF project No. 10C0457/11S163) conducted at dose levels of 0, 600 and 1000 mg/kg bw/d. In this study, a NOAEL was not established due to erosions and ulcerations in the stomach of different animals at both dose levels of 600 and 1000 mg/kg bw/d as well as clinical findings like poor general condition, piloerection and body weight loss after 1 week of treatment.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes, for at least 16 - 20 hours
- Section schedule rationale: random
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on workdays, daily on weekends and public holidays
- Cage side observations checked in table [No.1-2] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first administration, weekly thereafter
- Cage side observations checked in table [No.3] were included.

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, weekly thereafter with the following exceptions for females: 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.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, except during mating

WATER CONSUMPTION: Monitored by daily visual inspection

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (16-20h)
- How many animals: 5 per sex and group
- Parameters checked in table No. 5 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes (16-20h)
- How many animals: 5 per sex and group
- Parameters checked in table No. 6 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: day 31 (males), day 52 (females)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: yes, during collection
- How many animals: 5 per sex and group
- Parameters checked in table No. 7 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 3 days prior to necropsy (day 29 males, day 50 females)
- Dose groups that were examined: 5 animals per sex and group (only females with litter)
- Parameters checked in table No. 4 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, (see table 8)
HISTOPATHOLOGY: Yes (see table 9)
Other examinations:
Reproductive performance
Statistics:
See table No. 10.
Clinical signs:
no effects observed
Description (incidence and severity):
On study days 7 and 14 in one male animal of test group 2 (150 mg/kg bw/d) showed an encrusted eye during the detailed clinical observations (DCO).
The DCO on study days 0, 21, 28, 35 in male animals of test groups 0-3 (0, 50, 150 and 500 mg/kg bw/d) did not reveal any additional abnormalities.
In female animals of test groups 0-3 (0, 50, 150 and 500 mg/kg bw/d), DCO on study days 0, 7, 14, 21, 28, 35, 42, 49, 56 did not reveal any abnormalities.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal of test group 3 (500 mg/kg bw/d) was sacrificed in a moribund condition on gestation day 24.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related changes in body weight or body weight gain were observed for male and female animal of test groups when compared to control groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse findings were noted.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse findings were noted.
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 groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) large unstained cell (LUC) counts were lower compared to controls. Regarding this relatively short study period, only an increase but no decrease of the LUCs, which represent commonly immature cells, could have been related to a pathological effect. Therefore, this alteration was regarded as incidental and not treatment-related.
In females of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) relative monocyte counts and in females of test groups 1 and 3, only, absolute monocyte cell counts were higher compared to controls. Both parameters were not dose-dependently changed. Even in test
group 3 (500 mg/kg bw/d) this was an isolated finding with no change in other differential blood cell counts. Therefore, this alteration was 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 males of test group 3 (500 mg/kg bw/d), alanine aminotransferase (ALT) activities were decreased. The mean was 18% lower compared to that of the controls. There was no dosedependent decrease of the ALT activities. Therefore, this slight effect in test group 3 was regarded as incidental rather than treatment-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
In females of test group 2 (150 mg/kg bw/d), the urine pH value was higher compared to controls. This effect was not dose-dependently altered and, therefore, it was regarded as isolated, incidental and not treatment-related.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related effects were observed.
One female animal 6 of the control group showed increased response after audition and very frequent vocalizations when touched. A relation to treatment was excluded.
There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group. Regarding single intervals, an isolated significantly increased value was observed at interval 11 for male animals of test group 3 (500 mg/kg bw/d). The finding was considered to be incidental.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
All mean weight parameters (absolute and relative) did not show significant differences when compared to the control groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: General systemic toxicity
Key result
Critical effects observed:
no

Discussion

The test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 50, 150 and 500 mg/kg bw/d. The test substance is not classified for its skin/ eye irritant or sensitizing properties, although erythema and ear swelling were observed during the LLNA above a concentration of 10%.

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (50, 150 and 500 mg/kg bw/d) during the entire study period.

Salivation after treatment was seen in all animals of test group 3 (500 mg/kg bw/d) and sporadically in a few animals of test group 2 (150 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level of 500 mg/kg bw/d. The findings observed in test groups 1 (50 mg/kg bw/d) were assessed as being incidental and not related to treatment. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 500 mg/kg bw/d. Regarding pathology, all 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 this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of the test substance to male and female Wistar rats revealed no signs of systemic toxicity up to a dose level of 500 mg/kg bw/d in animals of both sexes. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 500 mg/kg bw/d in male and female animals.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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:
22 March 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA 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:
Lot/batch No.: 110007P040

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (male and female animals were derived from different litters to rule out mating of siblings)
- Age at study initiation: 10-11 wks
- Weight at study initiation: on average: Males: 335g; Females: 197g
- Fasting period before study: no
- Housing: single (except during mating and during lactation) in Markrolon type M III cages
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal ad lib.
- Water: ad lib.
- Acclimation period: app. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The desired amount of test substance was weighed, and corn oil was added up to the correct volume. To prepare a homogenous suspension, the mixture was stirred with a magnetic stirrer also during administration. The test substance preparations were produced at least once a week and were stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle: The test substance is poorly soluble in water, but forms a homogenous suspension in corn oil, which is also non-toxic to rats.
- Concentration in vehicle: 1.25; 3.75; 12.5 g/100mL
- Amount of vehicle: 4 mL/ kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1 over night
- Length of cohabitation: until sperm was detected in vaginal smear or for a maximum of 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90.0-110.0% of the nominal concentrations. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in corn oil.
Duration of treatment / exposure:
Males: 35 days
Females: 56 days
Frequency of treatment:
daily (except to animals being in labor)
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 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: The selection of doses was based on the results of a test study in female Wistar rats (BASF project No. 10C0457/11S163) conducted at dose levels of 0, 600 and 1000 mg/kg bw/d. In this study, a NOAEL was not established due to erosions and ulcerations in the stomach of different animals at both dose levels of 600 and 1000 mg/kg bw/d as well as clinical findings like poor general condition, piloerection and body weight loss after 1 week of treatment.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes, for at least 16 - 20 hours
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on workdays, daily on weekends and public holidays
- Cage side observations: check for moribund animals, pertinent behavioral changes, signs of overt toxicity, parturation, littering and lactation behavior of the dams
- Cage side observations checked in table No.1-2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first administration, weekly thereafter
- Cage side observations checked in table No.3 were included.

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, weekly thereafter with the following exceptions for females: 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.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, except during mating

WATER CONSUMPTION: Monitored by daily visual inspection

OTHER:
- Functional observation battery and motor activity measurement of 5 males and females per group 2 days prior to sacrifice (Parameters checked in table No. 4 were examined)
- Urinanalysis in 5 males and females per group one day prior to necropsy (Parameters checked in table No. 7 were examined.)
- Clinicochemical and hematological examinations 5 males and females on the day of necropsy after fasting for 16h (Parameters checked in table No. 5 and 6 were examined.)
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in male parental animals:
testis weight, epididymis weight, stages of spermatogenesis in the male gonads
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical symptoms, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, externally and organs were assessed macroscopically
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on day 36
- Maternal animals: All surviving animals on day 57

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations: yes

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 8-9 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
NECROPSY
- All surviving pubs were subjected to postmortem examinations: external examination and macroscopic examination of organs. Animals with notabel findings or abnormalitites were evaluated on a case-by-case basis.
Statistics:
See table No. 10
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 (vaginal sperm detected in females) / number of males placed with females x100
Male fertility index (%) = number of males proving their fertility (implants in utero) / number of males placed with females x100

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 (vaginal sperm detected) / number of females placed with males x100
Female fertility index (%) = number of females pregnant (implants in utero) / number of females mated (vaginal sperm or implants in utero) x100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x100
Offspring viability indices:
Live birth index (%) = number of liveborn pups at birth / total number of pups born x100
Viability index (%) = number of live pups on day 4 after birth / number of live pups on the day of birth x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Almost all male and most female animals of the high dose group showed salivation within 2 hours after the administration on several days of the study. Salivation within 2 hours after treatment was also seen in several mid dose male and female animals. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract.

On study days 7 and 14 in one male animal of test group 2 (150 mg/kg bw/d) showed an encrusted eye during the detailed clinical observations (DCO). The DCO on study days 0, 21, 28, 35 in male animals of test groups 0-3 (0, 50, 150 and 500 mg/kg bw/d) did not reveal any additional abnormalities. In female animals of test groups 0-3 (0, 50, 150 and 500 mg/kg bw/d), DCO on study days 0, 7, 14, 21, 28, 35, 42, 49, 56 did not reveal any abnormalities.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal of test group 3 (500 mg/kg bw/d) was sacrificed in a moribund condition on gestation day 24.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related changes in body weight or body weight gain were observed for male and female animal of test groups when compared to control groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse findings were noted.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse findings were noted.
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 groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) large unstained cell (LUC) counts were lower compared to controls. Regarding this relatively short study period, only an increase but no decrease of the LUCs, which represent commonly immature cells, could have been related to a pathological effect. Therefore, this alteration was regarded as incidental and not treatment-related.
In females of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) relative monocyte counts and in females of test groups 1 and 3, only, absolute monocyte cell counts were higher compared to controls. Both parameters were not dose-dependently changed. Even in test
group 3 (500 mg/kg bw/d) this was an isolated finding with no change in other differential blood cell counts. Therefore, this alteration was 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 males of test group 3 (500 mg/kg bw/d), alanine aminotransferase (ALT) activities were decreased. The mean was 18% lower compared to that of the controls. There was no dosedependent decrease of the ALT activities. Therefore, this slight effect in test group 3 was regarded as incidental rather than treatment-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
In females of test group 2 (150 mg/kg bw/d), the urine pH value was higher compared to controls. This effect was not dose-dependently altered and, therefore, it was regarded as isolated, incidental and not treatment-related.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related effects were observed.
One female animal 6 of the control group showed increased response after audition and very frequent vocalizations when touched. A relation to treatment was excluded.
There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group. Regarding single intervals, an isolated significantly increased value was observed at interval 11 for male animals of test group 3 (500 mg/kg bw/d). The finding was considered to be incidental.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index was 100% in all test groups (0, 50, 150 and 500 mg/kg bw/d). The male fertility index ranged between 80% (500 mg/kg bw/d) and 90% (0, 50, 150 mg/kg bw/d).
The female mating index calculated after the mating period for F1 litter was 100% for all test groups (0, 50, 150 and 500 mg/kg bw/d). The female fertility index varied between 80% (500 mg/kg bw/d) and 90% (0, 50, 150 mg/kg bw/d).
The mean duration of gestation was between 22.1 and 22.6 days and did not show significant differences. Not all sperm-positive females delivered pups. Therefore, the gestation index reached 100% in test groups 0-2 and 88% in test group 3.
One high dose female animal, that was not pregnant, revealed a decrease in ovary size and a diffuse atrophy of both ovaries and no corpora lutea. This was regarded to be the cause of the infertility in this pair. All other females and their male mating partners did not show gross lesions in reproduction relevant organs which could explain the lack of offspring.

Details on results (P0)

ORGAN WEIGHTS (PARENTAL ANIMALS)
All mean weight parameters (absolute and relative) did not show significant differences when compared to the control groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
All findings were considered to be incidental or spontaneous in origin and without any relation to treatment.
The high dose female animal that was sacrificed in a moribund state revealed a decidual reaction with consequent inflammation of the uterus. Decidual reaction is a proliferation of decidual cells (tissue of endometrial origin lining of the uterus, which is in contact with the fetal membranes and the placental plate). They often are assiocated with inflammation as observed in this animal. The local inflammation in the uterus can lead to disturbance of the general condition or, if the inflammation is spreading, sepsis. This was regarded to be the reason for the moribund state of this animal but was not regarded to be treatment-related.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fertility and reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Ten of 14 pups in the litter of one female animal of test group 1 (50 mg/kg bw/d) showed reduced nutritional condition on PND 0. All pups in this litter died between PND 0- 1. Each one pup of two female animals of test group 2 (150 mg/kg bw/d) died on PND 0. These findings were assessed as being incidental as no dose-reponse relationship occurred. The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Single stillborn pubs were seen in the low dose and mid dose group only. The mean number of delivered pups per dam and the rate of liveborn and stillborn pups reflect the normal range of biological variation inherent in the strain used in this study.
The viability index as indicator for pup mortality between PND 0 and 4 was 89% in the low dose group (all pups of one female died), 99% in the mid dose group (1 pup of one female died) and 100% for high dose and control females. As the decreased viability index in the low dose group was still within the historical control data, statistically not significant, and related to mentioned litter loss of only one female animal, the finding was assessed to be incidental and not related to treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values. Each 1 runt was seen in test groups 1 (female pup; 50 mg/kg bw/d) and 2 (male pup; 150 mg/kg bw/d). Both values were within the range of the biological variation inherent in the strain of rats used for this study.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Post mortem autolysis was observed for several pups in all test groups including the control. For 11 pups of test group 1 (50 mg/kg bw/d) an empty stomach was observed. Three pups of test group 1 (50 mg/kg bw/d) were not assessed because they were cannibalized on PND 1. The findings were assessed as being spontaneous in nature and without biological relevance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion