Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-11-18 to 2005-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Version / remarks:
87/302/EEC
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Uvinul A Plus
- Physical state: Solidified melt/yellow
- Analytical purity: >=98.8%
- Lot/batch No.:
ad 1) Labor Jr Nr. 31 656/25-5 (from Nov. 25, 2003 - Jul. 22, 2004)
ad 2) 301 76216K0 (from Jul. 22, 2004 until the scheduled termination of the in life part of the study)
- Date of production:
ad 1) July 24, 2003
ad 2) January 1,2004
- Stability under test conditions:
ad 1) The stability of batch No. Labor Jr Nr. 31656/25-5 under storage conditions was confirmed by reanalysis (analytical report 04L001 89).
ad 2) The stability of batch No. 30176216K0 under storage conditions over the exposure period was guaranteed (expiry date Dec. 26, 2004, analytical report 04L00099); however, stability was additionally verified by reanalysis (analytical report 05L00039).
- Storage condition of test material: Room temperature, dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Wistar rats Crl:WI(Han) (former: CrlGIxBrIHan:WI)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (former name: Charles River Laboratories, Germany)
- Age at study initiation: (P) 28-35 days (arrival); male: 35+/-1 days and female 42+/-1 days (beginning of treatment)
- Weight at study initiation: (P) Males: 152.2 (137.1-170.2) g; Females: 97.8 (83.2-107.9) g;
- Housing:
During the study period, the rats were housed individually in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, Germany (floor area of about 800 cm2 ), with the following exceptions:
* overnight matings: male and female mating partners housed together in type DK III cages
* gestation day 18 - lactation day 21: pregnant animals and their litters housed in Makrolon type M III cages.
The M III cages were also supplied by BECKER & CO. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet (e.g. ad libitum): The food used was ground Kliba maintenance diet mouse/rat "GLP", meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day of or the day before necropsy).
- Water (e.g. ad libitum): Drinking water was supplied from water bottles (ad libitum).
- Acclimation period: 1 week (2003-11-18 to 2003-11-25)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 2003-11-18 To: 2005-06-02

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Due to the aggregate state of Uvinul A Plus (solidified melt), the test substance was melted at 70 degree Celsius and homogenized by shaking before weighing out in a beaker and preparing the mixtures of food and test substance.
Acetonic solutions (approx. 100 mL Acetone) of the respective concentrations were made.
These solutions were sprayed on about 1.5 kg diet in a rotation vaporizer (Heidolph VV220).
Acetone was removed by heating up to about 40 degree Celsius. Thereafter these premixes were adjusted to the desired concentrations with appropriate amounts of food and mixed for about 10 minutes in a laboratory mixer (Ruberg Mischtechnik KG, Paderborn, Germany).

DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets were prepared at intervals, which guaranteed that the concentration of test substance in the diet remained stable throughout the feeding period.
- Storage temperature of food: Room temperature, dark

VEHICLE
For details see preparation of dosing solutions.
Details on mating procedure:
- Males/Females ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "day 0" and the following day "day 1" p.c. (post coitum).
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF Aktiengesellschaft, Ludwigshafen, Germany.
The stability of the test substance in the diet over 49 days at room temperature was investigated analytically. Homogeneity and concentration control analyses had been carried out at the beginning and toward the end of the premating periods, as well as during the periods after weaning and during the rearing period. At least one analysis of the test substance preparations of the female animals had been carried out during the gestation and lactation periods. Details of the analytical time scheduled are provided in the raw data. Of each sample one additional reserve sample had been taken.
Duration of treatment / exposure:
F0 male: from 2003-11-25 to 2004-03-29 or 2004-03-31
F0 female: from 2003-11-25 to 2004-04-04
F1 male: from 2004-03-30 to 2004-08-03 or 2004-08-05
F1 female: from 2004-03-30 to 2004-08-09
Frequency of treatment:
continuous via the diet
Details on study schedule:
- F1 parental animals not mated until 75 days after selected from the F1 litters.
- Selection of parents from F1 generation: after weaning
- Age at mating of the mated animals in the study: F0: male 35 d + 75 d; female 42 d + 75 d F1: 21 to 30 d + 75 d.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
F0 generation: in total 100 animals of each sex; 25 animals per sex per dose group.

After weaning, 25 males and 25 or 24 females* of the F1 pups of all test groups (00, 01, 02 and 03) were taken per group as the basis of the F1 generation parental animals. These animals were chosen by lot during rearing; it was attempted to take each litter into account. If fewer than 25 litters in these groups were available for selection or if one sex was missing in a litter, more animals were taken from different litters from the relevant test group to give the full number.

* One male F1 pup of the F0 dams (pup No. 8 from dam No. 110), which was selected to become a F1 parental rat, was erroneously declared as a female for further rearing. After this error had been detected during the rearing phase post weaning, this rat was sacrificed. Therefore, the F1 parental control group consisted of 24 F1 females, only.


Control animals:
yes, plain diet
Positive control:
no positive control

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All parental animals were checked daily for overt signs of toxicity. The littering and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only special findings (e.g., animal could not litter, umbilical cord not cut) were documented on an individual dam basis. The littering behavior of the dams was also inspected on weekdays (except public holidays) in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the 24-hour period from about 3.00 p.m. of one day until about 3.00 p.m. of the following day. Deviations from this procedure were possible on Saturdays, Sundays and on public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations:
In general, the body weight of the male and female parental animals was determined on the first day of the premating period and then once a week at the same time of the day (in the morning); if possible, the weighings were carried out until the end of the study. The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
a) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 post coitum.
b) Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval.
c) Females with litter were weighed on the day after parturition (day 1 p.p.) and on days 4, 7, 14 and 21 post partum.
d) Females without litter were not weighed during the lactation phase.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual food consumption was determined once a week (each time for a period of at least 6 days) for the F0 and F1 parental animals. After the 10th (F0 and F1 generation parental animals) test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for days 0-7, 7-14 and 14-20 p.c. During the lactation period (animals with litter) food consumption was determined weekly for days 1-4, 4-7 and 7-14 p.p. Food consumption was not determined between days 14 and 21 after parturition as required in the test guidelines cited under 2.3., since during this time pups will begin to consume considerable amounts of solid food offered, and therefore there was no point in such measurement. Furthermore, there was no determination of food consumption in the females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).
Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluated daily for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating and were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at necropsy a vaginal smear was examined to determine the stage of the estrous cycle for each F0 and F1 female with scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in males:
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from the F0 and F1 males of all dose groups.
The following parameters were determined:
* sperm motility
* sperm morphology
* sperm head count (cauda epididymis)
* sperm head count (testis)
Sperm motility examinations were carried out in all groups in a randomized sequence. Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose group, only.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
On day 4 p.p., the individual litters were in general standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If it was not possible in single litters to have 4 pups/sex, it was proceeded in such a way that 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Standardization of litters was not performed in litters with 8 pups or less.


PARAMETERS EXAMINED
The following parameters were examined in offspring:
All pups derived from the F0 parents (F1 litter) and the F1 parents (F2 litter) were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter. Pups, which died before the first determination of their status on the day of birth, were designated as stillborn pups.
Parameters: pup viability/mortality; sex ratio; pup clinical observations; pup body weight; pup organ weights; pup necroscopy observations.


GROSS EXAMINATION OF DEAD PUPS:
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 described as follows. All pups with scheduled sacrifice (i.e. pups, which were culled on day 4 p.p., and pups, which were sacrificed on day 21 after birth or subsequent days) were killed by means of CO2. These pups were examined externally and eviscerated, their organs were assessed macroscopically. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1 - 4, 5 - 7, 8 - 14 and 15 - 21 of the lactation period were determined; however, 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, and on lactation days 4, 7, 14 and 21.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals - all surviving animals: After weaning of F1 pups the F0 generation male animals were sacrificed. After the F2 generation pups had been weaned, the F1 generation male animals were sacrificed.
- Female animals - all surviving animals: After weaning of F1 pups the F0 generation female animals were sacrificed. After the F2 generation pups had been weaned, the F1 generation female animals were sacrificed.


GROSS NECROPSY
All F0 and F1 generation parental animals were sacrificed by decapitation under CO2-anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The animal that died intercurrently was necropsied as soon as possible after death and assessed by gross pathology.


HISTOPATHOLOGY / ORGAN WEIGHTS
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined:
1. Anesthetized animals
2. Liver
3. Kidneys
4. Adrenal glands
5. Testes
6. Epididymides
7. Cauda epididymis
8. Prostate
9. Seminal vesicle with coagulation gland
10. Ovaries
11. Uterus
12. Spleen
13. Brain
14. Pituitary gland
15. Thyroid gland (with parathyroid gland)

The following organs of all F0 and F1 generation parental animals were fixed in neutral buffered 4% formaldehyde solution or in Bouin's solution, respectively:
1. Vagina
2. Cervixuteri
3. Uterus
4. Ovaries (fixed in Bouin's solution)
5. Oviducts
6. Left testis (fixed in Bouin's solution)
7. Left epididymis (fixed in Bouin's solution)
8. Seminal vesicle
9. Coagulation gland
10. Prostate
11. Pituitary gland
12. Adrenal glands
13. All gross lesions
14. Liver
15. Kidneys
16. Spleen
17. Brain
18. Thyroid gland (with parathyroid gland)

Differential Ovarian Follicle Count (DOFC):
Both ovaries of each animal were embedded in the same way one upon another in one paraplast block ("ovary 1" and "ovary 2"). "Ovary 1" referred to the lower ovary on the slide when the label of it showed to the right side under the microscope, and "ovary 2" indicated the upper ovary on the same slide, respectively. 10 ovarian sections (5 out of each side of the ovaries) were prepared at least 100 pm apart from the inner third of each ovary and evaluated.
Primordial follicles and growing follicles were counted according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press)
Postmortem examinations (offspring):
Pup necropsy observations
All pups with scheduled sacrifice (i.e. pups, which were culled on day 4 p.p., and pups, which were sacrificed on day 21 after birth or subsequent days) were killed by means of CO2. These pups were examined externally and eviscerated, their organs were assessed macroscopically. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Pup organ weights
After scheduled sacrifice brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and first female pup/litter were taken for these determinations. For the calculation of the respective relative organ weights, the pup body weights, determined routinely during the in-life phase on day 21 p.p., were taken.
Statistics:
Parameter
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), estrous cycle duration, number of mating days, duration of gestation, number of pups delivered per litter, duration of sexual, maturation (days to preputial separation, days to vaginal opening)
Statistical test
DUNNETT-test

Parameter
Male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy, sexual maturation data (preputial separation, vaginal opening), males with >4% abnormal sperm
Statistical test
FISHER'S EXACT test

Parameter
Proportions of affected pups per litter with necropsy observations
Statistical test
WILCOXON-test

Parameter
Total spermatids/g testis, total sperm/g cauda epididymides
Statistical test
WILCOXON-test

Parameter
% mortality
Statistical test
WILCOXON-test

Parameter
Pup organ weights (absolute and relative)
Statistical test
KRUSKAL-WALLIS test
post-test: WILCOXON-test

Parameter
Weight parameters
Statistical test
KRUSKAL-WALLIS test
post-test: WILCOXON-test

Parameter
Follicles: primordial, growing and primordial growing
Statistical test
WILCOXON-test
Reproductive indices:
Male:
For the males, mating and fertility indices were calculated for F1 and F2 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

Female:
The mating partners, the number of mating days until vaginal sperm could be detected, and gestational status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for FI and F2 litters according to the following formulas:

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

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

Gestation Index (%) = number of females with live pups an the day of birth/ number of females pregnant* x 100
* defined as the number of females with implants in utero
Offspring viability indices:
Viability and lactation indices were calculated according to the following formulas:
Viability Index (%) = number of live pups an day 4 after birth/number of live pups an the day of birth x 100
Lactation index (%) = number of live pups an day 21 after birth / number of live pups an day 4 after birth x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0 animals:
1000 mg/kg bw/day:
- Signs of an impaired general state in several parental rats.
300 mg/kg bw/day:
- Signs of an impaired general state in 2 parental females indicated by urine smeared fur during the last premating weeks
100 mg/kg bw/day:
- No test substance related findings.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0 animals:
1000 mg/kg bw/day:
- Statistically significantly reduced food consumption during the different phases of the study with values between 11% and 28% below controls.
- Statistically significantly decreased mean body weights (final weight 14% below controls) and impaired body weight gains (about 22% below controls if calculated for weeks 0-17) in the male rats
- Statistically significantly decreased mean body weights during premating (11% below controls at week 10), gestation (24% below controls on day 20 p.c.) and lactation (10% below controls on day 21 p.p.), as well as decreased body weight gain during premating (19% below controls if calculated for weeks 0-10), and gestation (51% below controls if calculated for days 0-20 p.c.) in female rats
300 mg/kg bw/day:
- Statistically significantly decreased mean body weight in the male rats (about 5% below controls) at study week 17 and impaired body weight gains in the male rats (about 7% below controls if calculated for weeks 0-17)
100 mg/kg bw/day:
- No test substance related findings.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
see remarks on results

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0 animals:
- No test substance related findings.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0 animals:
- No test substance related findings.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0 animals:
1000 mg/kg bw/day:
- 3 F0 dams with no delivery of F1 pups, but with dead implants in utero; consequently the gestation Index was decreased (83% versus 100% in the controls) and the postimplantation loss value was statistically significantly increased (22.1% versus 4.4% in the controls)
- Statistically significantly reduced number of implantation sites per dam (9.6 versus 11.6 in the controls)
- Statistically significantly reduced number of delivered pups per dam (8.8 versus 11 .0 in the controls)
300 mg/kg bw/day:
- No test substance related findings.
100 mg/kg bw/day:
- No test substance related findings.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 animals:
- No test substance related findings.

GROSS PATHOLOGY (PARENTAL ANIMALS)
F0 animals:
- No test substance related findings.

HISTOPATHOLOGY (PARENTAL ANIMALS)

F0 animals:
- No test substance related findings

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: based on clinical signs and reduction in food consumption and body weights (observed in both parental generations)
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: based on lower gestation index, increased post implantation loss, reduced mean number of implantation sites and reduced number of delivered pups secondary to maternal homeostasis.

Results: P1 (second parental generation)

Details on results (P1)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

F1 animals:
1000 mg/kg bw/day:
- Signs of an impaired general state in several parental females.
300 mg/kg bw/day:
- No test substance related findings.
100 mg/kg bw/day:
- No test substance related findings.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F1 animals:
1000 mg/kg bw/day:
- Statistically significantly reduced food consumption during the different phases of the study with values between 15% and 27% below controls.
- Statistically significantly decreased mean body weights (final weight 24% below controls) and impaired body weight gains (about 25% below controls if calculated for weeks 0-18) in the male rats
- Statistically significantly decreased mean body weights during premating (16% below controls at week 10), gestation (25% below controls on day 20 p.c.) and lactation (17% below controls on day 21 p.p.), as well as decreased body weight gain during premating (13% below controls if calculated for weeks 0-10), and gestation (40% below controls if calculated for days 0-20 p.c.) in female rats
300 mg/kg bw/day:
- Statistically significantly reduced food consumption in the males on several study weeks (starting at study week 10-11); if calculated for weeks 0-18, the respective value was about 4% below controls
- Statistically significantly decreased mean body weights in the male rats from study week 8 onwards until termination (final weight about 8% below controls) and impaired body weight gains in the male rats during weeks 6-8, 9-10 and 0-18 (about 8% below controls if calculated for weeks 0-18)
100 mg/kg bw/day:
- Statistically significantly reduced food consumption in the males on several study weeks (starting at study week 6-7); if calculated for weeks 0-18, the respective value was about 5% below controls
- Statistically significantly decreased mean body weights in the male rats from study week 8 onwards until termination (final weight about 6% below controls) and impaired body weight gains in the male rats during weeks 6-7 and 0-18 (about 6% below controls if calculated for weeks 0-18)

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
see remarks on results


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F1 animals:
- No test substance related findings.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F1 animals:
- No test substance related findings.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F1 animals:
1000 mg/kg bw/day:
- Reduced number of implantation sites per dam (9.4 versus 10.8 in the controls; without attaining statistical significance)
- Reduced number of delivered pups per dam (8.8 versus 10.1 in the controls; without attaining statistical significance)
300 mg/kg bw/day:
- No test substance related findings.
100 mg/kg bw/day:
- No test substance related findings.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F1 animals:
1000 mg/kg bw/day:
- statistically significant increased absolute/relative kidney weights in males (+16.7%/+64.3%)
- Other findings were not considered as test substance related.
300 mg/kg bw/day:
- No test substance related findings.
100 mg/kg bw/day:
- No test substance related findings.

GROSS PATHOLOGY (PARENTAL ANIMALS)
F1 animals:
- No test substance related findings.

HISTOPATHOLOGY (PARENTAL ANIMALS)
F1 animals:
1000 mg/kg bw/day:
- Slightly higher amounts of eosinophilic droplets in the tubular epithelial cells (identified as alpha2u-globuline) of the males
300 mg/kg bw/day:
- Slightly higher amounts of eosinophilic droplets in the tubular epithelial cells (identified as alpha2u-globuline) of the males
100 mg/kg bw/day:
- No test substance related findings.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: general toxicity based on clinical signs and reduction in food consumption and body weights (observed in both parental generations)
Dose descriptor:
LOEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: based on borderline reduction in food consumption and body weights in one paternal generation only
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: based on lower gestation index, increased post implantation loss, reduced mean number of implantation sites and reduced number of delivered pups secondary to maternal homeostasis.

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
F1 pups:
1000 mg/kg bw/day:
- Statistically significantly lower number of surviving pups during the early postnatal phase indicated by a statistically significantly lower viability index (95% versus 99% in the controls)
300 mg/kg bw/day:
- No test substance related findings.
100 mg/kg bw/day:
- No test substance related findings.

CLINICAL SIGNS (OFFSPRING)
F1 pups:
- No test substance related findings.

BODY WEIGHT (OFFSPRING)
F1 pups:
1000 mg/kg bw/day:
- Statistically significantly decreased mean body weights (24% below controls at weaning) and body weight gains (25% below controls, postnatal days 4-21) of F1 male and female pups
300 mg/kg bw/day:
- No test substance related findings.
100 mg/kg bw/day:
- No test substance related findings.

SEXUAL MATURATION (OFFSPRING)
F1 pups:
1000 mg/kg bw/day:
- Slight delays of sexual maturation in males and females secondary to the general delay of pup development
300 mg/kg bw/day:
- No test substance related findings
100 mg/kg bw/day:
- No test substance related findings.

ORGAN WEIGHTS (OFFSPRING)
F1 pups:
1000 mg/kg bw/day:
- absolute/relative pup organ weight changes (both sexes combined) in brain (-6%/+23%) , thymus (-25%/ not siginficant) and spleen (-45%/-28%)
300 mg/kg bw/day: (considered as chance findings)
- absolute/relative pup organ weight changes (both sexes combined) in spleen (not significant/-7%)
- mean absolute/relative spleen weights were slightly, but statistically significantly decreased in males.
- mean relative thymus weights were statistically significantly increased in the females.
100 mg/kg bw/day: (considered as chance findings)
- mean absolute/ relative spleen weights were slightly, but statistically significantly decreased in the females.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: based on growth and development of offspring

Results: F2 generation

Details on results (F2)

VIABILITY (OFFSPRING)
F2 pups:
1000 mg/kg bw/day:
- Statistically significantly lower number of surviving pups until weaning indicated by a statistically significantly lower viability index (85% versus 99% in the controls) and a statistically significantly lower lactation index (97% versus 100% in the controls)
300 mg/kg bw/day:
- No test substance related findings.
100 mg/kg bw/day:
- No test substance related findings.

CLINICAL SIGNS (OFFSPRING)
F2 pups:
- No test substance related findings.

BODY WEIGHT (OFFSPRING)
F2 pups:
1000 mg/kg bw/day:
- Statistically significantly decreased mean body weights (26% below controls at weaning) and body weight gain (28% below controls, postnatal days 4-21) of F2 male and female pups
300 mg/kg bw/day:
- Statistically significantly decreased mean body weights on day 21 p.p. (about 6% below controls) and statistically significantly impaired body weight gain on days 14-21 and 4- 21 p.p. (about 7% below controls, postnatal days 4-21) of F2 male and female pups
100 mg/kg bw/day:
- No test substance related findings.

ORGAN WEIGHTS (OFFSPRING)
F2 pups:
1000 mg/kg bw/day:
- absolute/relative pup organ weight changes (both sexes combined) in brain (-7%/+23%) , thymus (-24%/ not siginficant) and spleen (-46%/-28%)
300 mg/kg bw/day:
- Mean absolute spleen weights, were marginally, but statistically significantly decreased in the females.
100 mg/kg bw/day:
- No test substance related findings.

Effect levels (F2)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: based on growth and development of offspring

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

 

 

         Dose group (mg/kg bw/d)

 

 

100

300

1000

F0

male

101.3

303.8

1010.3

 

female premating

103.7

311.4

1033

 

female gestation

103.4

312

927.8

 

female lactation

98.7

294

952.4

 

 

 

 

 

F1

male

101.1

303.2

1009.9

 

female premating

102

305.5

1017.1

 

female gestation

101.2

301.7

955.2

 

female lactation

93.6

281.1

902.5

 

Applicant's summary and conclusion