Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Di-(2-ethylhexyl)amine
- Physical state: Colourless, clear liquid
- Analytical purity: 98.5 g/100 g
- Lot/batch No.: 000STD77L0
- BASF test substance number: 11/0454-1
- Expiration date of the lot/batch: 11.11.2013
- Stability of Test Item in Vehicle: 8 days at room temperature
- Storage condition of test material: Room temperature (20 ± 5 °C)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 293 to 336 g, Females: 205 to 250 g
- Housing: In groups of five or six in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C rodent maintenance diet (Provimi Kliba SA, Kaiseraugst/Switzerland) was available ad libitum.
- Water: Community tap-water from Fuellinsdorf was available ad libitum in water bottles.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. Di-(2-ethylhexyl)amine was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as [day 0] of pregnancy
- Further matings after two unsuccessful attempts: no (all animals were mated within the first pairing period)
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group (middle only) as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle to confirm the stability. On 16-Aug-2012, samples were taken from the middle to confirm concentration. The samples were analyzed by GC coupled to an FID detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Duplicates were taken of all samples. The results indicate the accurate use of the test item di-(2-ethylhexyl)amine and corn oil as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
- Males: minimum 4 weeks;
- Females: approximately 6 weeks;
Males were sacrificed after treatment of 28 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 5 post partum. Since in several females a birth did not occur on the expected date (day 21 post coitum), these dams were sacrificed on day 25 post coitum.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
7.5 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day range-finding study (D52701, BASF project number 01R0454/11X312) the dose levels of 7.5, 25, and 75 mg/kg bw per day were selected for the present study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Viability / Mortality: twice daily; Clinical signs: once daily, during acclimatization and up to day of necropsy; additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing;
- Cage side observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern); changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior;

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration of the test item (day 6 of acclimatization) and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena;

BODY WEIGHT: Yes
- Time schedule for examinations: daily (from treatment start to day of necropsy)

FOOD CONSUMPTION AND COMPOUND INTAKE: Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period days 1 - 8 and 8 - 11; Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 - 14 and 14 – 21 and days 1 - 4 of the lactation; no food consumption was recorded during the pairing period;
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

OTHER: see Repeated Dose Toxicity
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
macroscopic and microscopic evaluation of testis (including sperm staging), epididymis, prostate and seminal vesicles with coagulating glands;
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, presence of gross anomalies, pups were weighed individually on days 1 and 4 post partum]

GROSS EXAMINATION OF DEAD PUPS:
yes, external examinations
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals (after treatment of 28 days, when no longer needed for the assessment of reproductive effects)
- Maternal animals: All surviving animals (dams were sacrificed on day 5 post partum; since in several females a birth did not occur on the expected date (day 21 post coitum), these dams were sacrificed on day 25 post coitum)

GROSS NECROPSY
- Gross necropsy: For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of apparently non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
(all gross lesions, testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group, the remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically; special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure; histological examination of ovaries was carried out on the females that did not give birth; in addition, microscopic examination of the reproductive organs of all infertile males was made;)

Organ weights:
- all parental males: testes and epididymides (of all parental males);
- from 5 males and 5 females killed at the end of the study which were selected for hematology and clinical chemistry examination from each group: adrenal glands (weighed as pairs), brain, heart, kidneys (weighed as pairs), uterus (including cervix), prostate, liver, thymus, spleen, thyroid (after fixation), ovaries (weighed as pairs), seminal vesicles (inclusive coagulating gland);

Tissue preservation:
- all parental males: prostate, seminal vesicles with coagulating gland, testes (in Bouin’s fixative), epididymides (in Bouin’s fixative);
- all parental females: ovaries (with oviduct), uterus (with vagina);
- all males and females: gross lesions, brain, spinal chord (cervical, thoracic, lumbar), small and large intestines (incl. Peyer’s patches), stomach (forestomach and glandular stomach), liver, kidneys, adrenals, lymph nodes (axillary and mesenteric), urinary bladder, heart, thymus, thyroids and parathyroids, trachea and lungs, spleen, peripheral nerve (sciatic), bone marrow (femur);
- all males and females (only examined by histopathology in case of macroscopic findings indicative of potential toxicity): aorta, eyes with optic nerve and harderian gland, lacrimal gland, larynx, nasal cavity, esophagus, pituitary gland, femur with knee joint, mammary gland (male and female), pancreas, salivary glands – mandibular, sublingual, skeletal muscle, sternum with bone marrow, pharynx;
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on day 4 post partum.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations
Statistics:
- food consumption, body and organ weights, clinical laboratory and reproduction data and macroscopical findings: means and standard deviations of various data were calculated; the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex; the Steel-test (many-one rank test) was applied instead of the
Dunnett-test when the data could not be assumed to follow a normal distribution; Fisher's exact-test was applied if the variables could be dichotomized without loss of information;
Reproductive indices:
Calculation of:
Percentage mating = ( Females mated / Females paired) * 100
Fertility index = ( Females achieving a pregnancy / Females paired) * 100
Conception rate = ( Females achieving a pregnancy / Females mated) * 100
Gestation index = ( Number of females with living pups / Females pregnant) * 100
Offspring viability indices:
Birth index = (pups born alive / number of implantations) * 100
Viability index = (pups alive before culling on day 4 p.p. / pups born alive) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were noted in controls or in animals treated at 7.5 and 25 mg/kg bw/day. At 75 mg/kg bw/day in males and females, bedding in mouth were observed from the end of the pre-pairing period or pairing period onwards until one day before necropsy. This was accompanied by slight salivation in some animals. This was considered to be a sign of discomfort and without toxicological relevance. No findings were noted at detailed weekly clinical observation.
Mortality:
no mortality observed
Description (incidence):
There were no unplanned deaths during the whole course of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related effects on mean body weights and mean body weight gain were noted in males at 7.5 and 25 mg/kg bw/day. At 75 mg/kg bw/day, there was a slight decrease in mean body weight gain after treatment start during the first week of the pre-pairing period (23 g compared to 28 g in the control group). Afterwards no decrease was observed anymore. This slight effect could be test item-related but was considered to be not adverse. The overall differences in mean body weight gain at the dose levels of 0, 7.5, 25 and 75 mg/kg bw/day were: +17%, +15%, +17% and +16% during the pre-pairing period and +3%, +2%, +3% and +2% during the after pairing period (percentages refer to the body weight gain within the period). No test item-related effects on mean body weight and mean body weight gain were noted in females. The overall differences in mean body weight gain at the dose levels of 0, 7.5, 25 and 75 mg/kg bw/day were: +7%, +8%, +8% and +7% during the pre-pairing period, +50%, +52%, +53% and +50% during the gestation period and +5%, +3%, +6% and +4% during the lactation period (percentages refer to the body weight gain within the period).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related effects on mean food consumption were noted in males and females.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The assessment of the hematology data did not reveal any test item-related effects in males and females. Statistically significant differences (lower hemoglobin, MCHC, and eosinophil values in females at 7.5 mg/kg bw/day; lower eosinophil values in females and higher RDW levels in males at 25 mg/kg bw/day; higher HDW values in females at 75 mg/kg bw/day) showed either no dose-dependency or the values were in the range of the historical control data.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The assessment of the clinical biochemistry data did not reveal any test item-related effects in males and females. At 75 mg/kg bw/day, there was an increase in triglycerides and a decrease of total bilirubin in males and an increase in total cholesterol in females. The values of total bilirubin were within the historical control data. The alterations of triglycerides and cholesterol were considered to be of no toxicological relevance, since the differences to the control group were minor and were evident in one sex only. The same applies to the significantly higher ALP activities in females at 7.5 mg/kg bw/day, since there was no dose-dependency.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related findings were noted during the functional observational battery in males and females at any dose level. One female treated at 7.5 mg/kg bw/day showed increased rearings. At 75 mg/kg bw/day, one female vocalized spontaneously when held in the hand. These isolated findings were considered to be incidental. Another female at 75 mg/kg bw/day showed salivation, which was already noted and discussed at the daily clinical investigation. The body temperature in males treated at 25 and 75 mg/kg bw/day was statistically significantly decreased (38.1°C and 37.6°C, respectively, compared to 38.6°C in the control group). Since these values were within the range of the historical control data (37.5 - 38.6°C), this finding was considered to be not test item-related. No such trend towards lower body temperatures was noted in females. Locomotor activity was not affected by the treatment with the test item in males or females at any dose level.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive performance:
no effects observed
Description (incidence and severity):
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 2.9, 2.8, 2.7 and 2.7 days at the dose level 0, 7.5, 25 and 75 mg/kg bw/day, respectively. The median precoital time was 3 days in all groups. One female each in every dose level was not pregnant. As a result the fertility index in all groups was 90.9%.
Mean number of corpora lutea per dam (determined at necropsy) was not affected by treatment with the test item (14.7, 15.0, 14.6 and 14.1 in order of ascending dose level).
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.5, 21.4, 21.5 and 21.6 days, in order of ascending dose level.
No effects on implantation rate or post-implantation loss were observed at any dose level when compared with controls. The mean number of implantations per dam was 13.1, 13.3, 13.2 and 12.3 in order of ascending dose levels. The mean incidence of post-implantation loss as a percentage of total implantations was 8.4%, 10.5%, 7.6% and 15.3%, in order of ascending dose level.
No effects on litter size were observed at any dose level. Mean litter size at first litter check was 12.1, 12.0, 12.2 and 10.4 pups in order of ascending dose levels. One dead pup at first litter check was recorded in the control group and at 7.5 mg/kg bw/day.
No effects on postnatal loss were observed at any dose level. At 75 mg/kg bw/day, two dams lost their whole litter between day 0-4 post partum. One of the litters consisted of only a single pup and it is a common finding that litters with single pups will be lost. The other litter contained 7 pups, out of these 7 pups two pups were injured at first litter check and all pups were missing on day 2 or 3 post partum. This finding was considered to be incidental due to its isolated occurrence.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
There were no unplanned deaths during the whole course of the study. No clinical signs were noted in controls or in animals treated at 7.5 and 25 mg/kg bw/day. At 75 mg/kg bw/day in males and females, bedding in mouth were observed from the end of the pre-pairing period or pairing period onwards until one day before necropsy. This was accompanied by slight salivation in some animals. This was considered to be a sign of discomfort and without toxicological relevance. No findings were noted at detailed weekly clinical observation.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
No test item-related effects on mean body weights and mean body weight gain were noted in males at 7.5 and 25 mg/kg bw/day. At 75 mg/kg bw/day, there was a slight decrease in mean body weight gain after treatment start during the first week of the pre-pairing period (23 g compared to 28 g in the control group). Afterwards no decrease was observed anymore. This slight effect could be test item-related but was considered to be not adverse. The overall differences in mean body weight gain at the dose levels of 0, 7.5, 25 and 75 mg/kg bw/day were: +17%, +15%, +17% and +16% during the pre-pairing period and +3%, +2%, +3% and +2% during the after pairing period (percentages refer to the body weight gain within the period).
No test item-related effects on mean body weight and mean body weight gain were noted in females. The overall differences in mean body weight gain at the dose levels of 0, 7.5, 25 and 75 mg/kg bw/day were: +7%, +8%, +8% and +7% during the pre-pairing period, +50%, +52%, +53% and +50% during the gestation period and +5%, +3%, +6% and +4% during the lactation period (percentages refer to the body weight gain within the period).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
No test item-related effects on mean food consumption were noted in males and females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 2.9, 2.8, 2.7 and 2.7 days at the dose level 0, 7.5, 25 and 75 mg/kg bw/day, respectively. The median precoital time was 3 days in all groups. One female each in every dose level was not pregnant. As a result the fertility index in all groups was 90.9%.
Mean number of corpora lutea per dam (determined at necropsy) was not affected by treatment with the test item (14.7, 15.0, 14.6 and 14.1 in order of ascending dose level).
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.5, 21.4, 21.5 and 21.6 days, in order of ascending dose level.
No effects on implantation rate or post-implantation loss were observed at any dose level when compared with controls. The mean number of implantations per dam was 13.1, 13.3, 13.2 and 12.3 in order of ascending dose levels. The mean incidence of post-implantation loss as a percentage of total implantations was 8.4%, 10.5%, 7.6% and 15.3%, in order of ascending dose level.
No effects on litter size were observed at any dose level. Mean litter size at first litter check was 12.1, 12.0, 12.2 and 10.4 pups in order of ascending dose levels. One dead pup at first litter check was recorded in the control group and at 7.5 mg/kg bw/day.
No effects on postnatal loss were observed at any dose level. At 75 mg/kg bw/day, two dams lost their whole litter between day 0-4 post partum. One of the litters consisted of only a single pup and it is a common finding that litters with single pups will be lost. The other litter contained 7 pups, out of these 7 pups two pups were injured at first litter check and all pups were missing on day 2 or 3 post partum. This finding was considered to be incidental due to its isolated occurrence.

ORGAN WEIGHTS (PARENTAL ANIMALS):
In males, no effects on organ weights were noted. In females, a dose-dependent and statistically significant increase in liver and kidney weight as
well as liver and kidney to body and to organ weight ratio were noted at 25 and 75 mg/kg bw/day. This was considered compound-related, but not adverse, since there was no evidence for an impaired organ function by clinical chemistry, macroscopic examinations at necropsy and histopathology.

GROSS PATHOLOGY (PARENTAL ANIMALS):
There were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded were considered to be within the range of normal background alterations. In males, incidental findings were a reddish or dark red discoloration of the pancreas and a yellowish and firm nodule in the pancreas. Additionally, two males showed following findings in the testes: at 25 mg/kg bw/day a hardening on the right side was noted, and at 75 mg/kg bw/day a reduction in size was present. However, those two males mated successfully. At microscopic examination of those two animals, the left testis of the male treated at 25 mg/kg bw/day was normal. In the male treated at 75 mg/kg bw/day, a moderate bilateral tubular degeneration/atrophy was noted at microscopic examination. Due to the low incidence and the lack of dose-dependency all these findings were considered to be incidental. In females, the most frequently incidental findings were a reddish or dark red discoloration of the ovaries and foci on or a reduction in size of the thymus, which was present in all dose levels without dose-dependency.

HISTOPATHOLOGY (PARENTAL ANIMALS):
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(systemic)
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOEL
Remarks:
(reproduction)
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
At 75 mg/kg bw/day, two pups from one litter showed a wound on the nose, and one of those two pups was also cold to the touch. By day three post partum, all pups from this litter were missing. These findings were considered not to be test item-related, since only one litter was affected. Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. The proportion of males at first litter check was 45, 48, 39 and 54%, in order of ascending dose level.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean pup weights on day 0 and day 1 post partum were not affected by treatment with the test item. On day 1 post partum mean pup weights were 6.2, 6.3, 6.3 and 6.1 g in order of ascending dose level. Also mean body weight gain was similar in all groups and on day 4 post partum and no effects on mean body weights were recorded.
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. The proportion of males at first litter check was 45, 48, 39 and 54%, in order of ascending dose level.
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings were noted at macroscopic examination of F1 pups.
Histopathological findings:
not examined
Other effects:
no effects observed

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
(developmental toxicity)
Generation:
F1
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 75 mg/kg body weight /day, because no adverse effects were observed.
Executive summary:

The purpose of this OECD 422 study was to generate preliminary information on the possible health hazards likely to arise from repeated exposure to Bis(2 -ethylhexyl)amine. In addition, it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Di-(2-ethylhexyl)amine was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

The following dose levels were applied: 0 (control group), 7.5, 25, or 75 mg/kg body weight/day.

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

The following results were obtained:

Parent animals

- General Tolerability: There were no unplanned deaths during the course of this study. Clinical signs were limited to animals treated at 75 mg/kg bw/day and consisted of bedding in mouth, accompanied by slight salivation in some isolated animals. This was considered to be a sign of discomfort and without toxicological relevance.

- Food consumption: No effects on food consumption of males and females were observed at any dose level.

- Body weights: At 75 mg/kg bw/day, there was a slight decrease in mean body weight gain after treatment start (23 g compared to 28 g in the control group). This slight effect could be test item-related but was

considered to be not adverse. In females no effects on mean body weight and mean body weight gain were noted.

- Clinical laboratory investigations: The assessment of the hematology and clinical biochemistry data did not reveal any test item related effects in males and females.

- Reproduction and breeding data: Mean precoital time, fertility index and conception rate were not affected by the treatment with the test item. Mean number of litter size was similar in all groups and no effects on postnatal loss were observed at any dose level.

- Organ weights: Liver and kidney weights were increased in females at 25 and 75 mg/kg bw/day. These findings were considered compound-related, but not adverse, since there was no evidence for an impaired organ function by clinical chemistry and histopathology.

- Macroscopical findings and histopathological examinations: There were no test item-related findings noted at necropsy. All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Litter data - F1 pups

- Findings at First Litter Check and during Lactation: No test item-related external findings were noted at first litter check.

- Pup Weights to Day 4 Post Partum: No effects on pup weight and pup weight gain were observed.

- Macroscopical Findings: At necropsy of pups, there were no abnormal findings.

Conclusion

The general NOAEL (No Observed Adverse Effect Level) was considered to be 75 mg/kg body weight/day.

The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 75 mg/kg body weight /day.