2-imidazolidone

Toxicity to reproduction

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

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-12 wks
- Weight at study initiation (mean): (P) Males: 316.6 g; Females: 210.6 g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4 (end of lactation).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Type NGM E-022) supplied by Abed® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: 1 week

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

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired weight, subsequently released with a magnetic stirrer. The test substance preparations were produced at least twice a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses of the test substance preparations: The analyses of the test substance preparations were carried out as a separate study at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study at a concentration of 5000 ppm. To verify the stability for the lowest concentration in the current study (100 ppm) a stability analysis was carried out during the study period. Concentration control Analysis of the test substance preparations were performed in samples of all concentrations at the start and towards the end of the administration period.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: from about 16.00 h until 07.00 - 09.00 h of the following morning, for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Duration of treatment / exposure:

Males were exposed for 30 days, females were exposed for 51 days

Frequency of treatment:

Daily

Duration of test:

52 days

Dose / conc.:

100 ppm (nominal)

Dose / conc.:

500 ppm (nominal)

Dose / conc.:

2 000 ppm (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: at the request of the sponsor
- Rationale for animal assignment: the animals were distributed according to weight among the individual test groups, separated by sex

Maternal examinations:

- Mortality: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.
- Clinical observations: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
- Detailed clinical observations: Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during “handling", fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine and pupil size.
- Water consumption: Generally, water consumption was determined once a week for male and female parental animals, with the following exceptions:
Water consumption was not determined during the mating period (male and female F0 animals), Water consumption of the F0 females with evidence of sperm was determined on GD 7, 14 and 20. And water consumption of F0 females, which gave birth to a litter was determined for PND 4.
Water consumption was not determined in 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) and in males after the premating period.
- Food consumption: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
Food consumption was not determined during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on GD 7, 14 and 20. Food consumption of F0 females, which gave birth to a litter was determined for PND 4. Food consumption was not determined in 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) and in males after the premating period.
- Body weight data: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 1) and on PND 4. Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Intake of test substance: The intake of test substance was calculated from the amount of water consumed and expressed in mg/kg body weight per day
- Functional observational battery: A functional observational battery (FOB) was performed in the first five parental males and females (with litter) per group and the groups were selected at the end of the administration period starting at about 10:00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Motor activity assessment: The motor activity assessment (MA) was carried out in the first five parental males and females (with litter) per group at the end of the administration period. Motor activity was measured on the same day as FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out starting at 14:00 h. On account of the measuring variant "staggered", the starting time varied according to the time needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements. After the transfer of the last animal the room of measurement was darkened. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.
- Male reproduction data: For the males, mating and fertility indices were calculated for F1 litters.
- Female reproduction and delivery data: For the females, mating, fertility and gestation indices were calculated for F1 litters.

Ovaries and uterine content:

Not determined because females were allowed to give birth.

Fetal examinations:

- Pup number and status at delivery: All pups delivered from the F0 parents 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 pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.
- Pup viability/mortality: 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.
- Pup clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Sex ratio: On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.
- Pup clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Pup body weight data: The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning).
- Pup necropsy observations: All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, viability index: Pairwise comparison of each dose group with the control group using FISHER'S EXACT-test for the hypothesis of equal proportions.
- Number of mating days: Pairwise comparison of the dose group with the control group using the WILCOXON-test (one-sided) with Bonferoni-Holm-Adjustment for the hypothesis of equal medians.
- Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
- Statistics of clinical pathology (except for urine color and turbidity): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- Statistics of pathology: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Indices:

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

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

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

Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
* defined as the number of females with implants in utero

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:

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

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

Post implantation loss (%) = (number of implantations - number of pups delivered/number of implantations) x 100

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:

Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x 100

Details on maternal toxic effects:

Details on maternal toxic effects:
CLINICAL EXAMINATIONS AND EXAMINATION OF REPRODUCTIVE PERFORMANCE: Parental clinical examinations and examination of reproductive performance:
- Mortality: No animal died prematurely in the present study.
- Detailed clinical observations: No abnormal clinical signs were observed.
- Clinical observations for females during gestation: One sperm-positive F0 female of test group 1 (No. 119) and two sperm-positive F0 female of the test group 3 (Nos. 134 and 138) did not deliver any F1 pups. No other clinical signs were observed.
- Clinical observations for females during lactation: One female animal of test group 3 showed complete litter loss. One female animal of test group 1 showed insufficient maternal care and did not nurse the pups properly during lactation period.
- Water consumption: Water consumption was significantly reduced in females of test group 3 (2000 ppm) on lactation day 4 (-37.9%).
- Food consumption: Food consumption during gestation was significantly decreased in females of test group 3 (2000 ppm) from study days 7-14 and 14-20 (up to -9.5%) and during the entire gestation period from day 0 to 20 (-8.8%). During lactation period the food consumption was significantly decreased (-40.2%) in females of test group 3 (2000 ppm).
- Body weight data: Body weight was significantly decreased in females of test group 3 (2000 ppm) on gestation day 20 (-6.2%) and during lactation days 1 and 4 (up to -9.9%). Body weight change value was significantly reduced in males of test group 3 (2000 ppm) from day 0 to 7 (-38.3%). Body weight change values were significantly reduced during gestation days 14-20 (-24.3%) and during the entire gestation period from day 0-20 in female animals of test group 3 (2000 ppm)

TEST SUBSTANCE INTAKE
The intake of the test substance (in mg/kg bw/d) was calculated on the basis of most recent individual body weights in each test group:
Test group 1 Test group 2 Test group 3
(100 ppm) (500 ppm) (2000 ppm)
F0 males (premating) 7.4 36.9 155.0
F0 females (premating) 10.2 47.8 191.0
F0 females (gestation) 11.0 53.1 222.0
F0 females (lactation) 17.0 86.6 234.2
- Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually: - Home cage observations: No test substance-related effects were observed, - Open field observations: No test substance-related effects were observed. - Sensorimotor tests/reflexes: No test substance-related effects were observed. - Quantitative Parameters: The values of landing foot splay test in males of test group 2 (500 ppm) were significantly increased (+27.4%). Due to the lack of dose response relationship this was assessed as being incidental.
- Motor activity measurement: Male animals of test group 1, 2 and 3 (100; 500 and 2000 ppm) showed a significantly lower value at interval 1 when compared to control males. Female animals of test group 3 (2000 ppm) showed a significantly lower value in interval 7. There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals. All described findings were assessed as being incidental and not related to treatment.

CLINICAL PATHOLOGY
- Hematology: No treatment-related, adverse changes among hematological parameters were observed. In females of test group 3 (2000 ppm) hemoglobin values were lower compared to controls. This was the only altered red blood cell parameter and the decrease was marginal (mean: -6.6%) although below the historical control range (hemoglobin 8.7-9.5 mmol/L). Therefore, this alteration was regarded as treatment-related but not adverse (Müller et al., 2006, ECETOC Technical Report No. 85, 2002).
- Clinical chemistry: No treatment-related changes among clinical chemistry parameters were observed. In females of test group 3 (2000 ppm), creatinine levels were higher compared to controls, but the mean was within the historical control range (creatinine 52.2-62.7 mmol/L) whereas the control mean was below the historical range. Creatinine was the only altered clinical chemistry parameter in this study. Therefore, this change was regarded as incidental and not treatment-related (ECETOC, Technical Report No. 85, 2002)
- Urinalyses: No treatment-related changes among urinalysis parameters were observed.

GROSS PATHOLOGY
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The female animals (Nos. 119, 134, 138), which were not pregnant as well as the male mating partners (Nos. 19, 34, 38) did not show relevant gross lesions.

HISTOPATHOLOGY
The number of animals with follicular hypertrophy/ hyperplasia was increased in males and females of test group 3 (2000 ppm). In affected animals the number of small follicles was increased or the follicular epithelium was higher, varying in size from cuboidal cells to columnar cells. The occurrence of hypertrophy/ hyperplasia in animals of test group 3 (2000 ppm) was considered to be treatment-related.
-Axillary lymph nodes and spleen: In the axillary lymph nodes of control males and females, most follicles were primary follicles; these are non-stimulated follicles without germinal centers. In some treated animals, the number of follicles with germinal centers (secondary follicles) was slightly (grade 2) or clearly (grade 4) increased. In the spleen of control male and female animals most follicles had no germinal centers. In some treated males and females the number of follicles with germinal centers was slightly (grade 2) or clearly (grade 4) increased when compared with control animals. The number of males and females with a higher number of secondary follicles (follicles with germinal centers) in the axillary lymph nodes or in the spleen was dose-related increased in all treatment groups. The increase of follicles with germinal centers was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
-Fertility: The female animals (Nos. 134, 138), which were not pregnant as well as the male mating partners (Nos. 34, 38) did not show histopathological findings explaining why the animals were not pregnant. From the female animal (No. 119) which was recorded as not pregnant as well as from the male mating partner (Nos. 19) only the axillary lymph node and the spleen were examined histologically, according to the study plan.

Key result

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Basis for effect level:

other: General toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- Pup viability/mortality: The viability index indicating pup mortality during lactation (PND 0 - 4) was between 98.5% (test group 0), 100% (test group 1), 98.4% (test group 2) and 81.6% (test group 3). For test group 0-2, these values were in the normal range of biological variation inherent in the strain of rats used for this study. For test group 3, a decreased pup viability was noted, which was also outside the historical control range. Although 7 to the 11 dead offsprings in this group came from only one litter (No. 135), a relationship to the treatment cannot be excluded.

Key result

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on decreased pup viability.

Key result

Dose descriptor:

NOAEL

Effect level:

37 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on decreased pup viability.

Key result

Dose descriptor:

NOAEL

Effect level:

57 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on decreased pup viability.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

ANALYSES

- Stability analysis: The stability of the test substance in drinking water was demonstrated over a period of 4 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.

- Concentration control analysis of the test substance preparations: 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 94.8 - 102% of the nominal concentrations. These results demonstrate the correctness of the concentrations of Ethylene Urea.

- Food analyses: On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1×105/g food. - Drinking water analyses: On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

- Bedding and enrichment analyses: On the basis of the analytical findings the bedding and the enrichment are found to be suitable. Levels given in Lab. Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum tolerable contaminants.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 11-0041
- Test substance No.: 08/0054-4

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-12 wks
- Weight at study initiation (mean): Males: 316.6 g; Females: 210.6 g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4 (end of lactation).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm²) and small amounts of bedding material.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Type NGM E-022) supplied by Abed® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum.
- Water (e.g. ad libitum): drinking water, ad libitum.
- Acclimation period: 1 week

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

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired weight, subsequently released with a magnetic stirrer. The test substance preparations were produced at least twice a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses of the test substance preparations: The analyses of the test substance preparations were carried out as a separate study at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study at a concentration of 5000 ppm. To verify the stability for the lowest concentration in the current study (100 ppm) a stability analysis was carried out during the study period. Concentration control analysis of the test substance preparations were performed in samples of all concentrations at the start and towards the end of the administration period.

Duration of treatment / exposure:

Males were exposed for 30 days, females were exposed for 51 days

Frequency of treatment:

Daily

Dose / conc.:

100 ppm

Remarks:

nominal in water

Dose / conc.:

500 ppm

Remarks:

nominal in water

Dose / conc.:

2 000 ppm

Remarks:

nominal in water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: at the request of the sponsor
- Rationale for animal assignment: the animals were distributed according to weight among the individual test groups, separated by sex.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked were: signs of morbidity, pertinent behavioral changes and signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period, on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION:
- Time schedule: food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 4.
Food consumption was not determined in 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) and in males after the premating period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week for male and female parental animals, with the following exceptions:
• Water consumption was not determined during the mating period (male and female F0 animals).
• Water consumption of the F0 females with evidence of sperm was determined on GD 7, 14 and 20.
• Water consumption of F0 females, which gave birth to a litter was determined for PND 4.
Water consumption was not determined in 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) and in males after the premating period.
The intake of test substance was calculated from the amount of water consumed and expressed in mg/kg body weight per day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning, at the end of the exposure period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked were: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning, at the end of the exposure period.
- Animals fasted: Yes
- How many animals: all
- Parameters checked were: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase), γ-Glutamyltransferase (GGT) (γ-glutamyl) peptide: aminoacid-γ-glutamyl-transferase), Sodium (Na), Potassium (K), Chloride (Cl), Inorganic phosphate (INP), Calcium (Ca), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG)

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the exposure period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked were: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in the first five parental males and females (with litter) per group and the groups were selected at the end of the administration period starting at about 10:00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations - home cage, open field and sensorimotor tests/reflexes - were performed at random.
The motor activity assessment (MA) was carried out in the first five parental males and females (with litter) per group at the end of the administration period. Motor activity was measured on the same day as FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out starting at 14:00 h. On account of the measuring variant "staggered", the starting time varied according to the time needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements. After the transfer of the last animal the room of measurement was darkened.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Duodenum, Epididymides, Testes, Thyroid glands.
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus.

HISTOPATHOLOGY: Yes
The following tissues were prepared for microscopic examination and weighed: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating gland, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal glands, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, viability index: Pairwise comparison of each dose group with the control group using FISHER'S EXACT-test for the hypothesis of equal proportions.
- Number of mating days: Pairwise comparison of the dose group with the control group using the WILCOXON-test (one-sided) with Bonferoni-Holm-Adjustment for the hypothesis of equal medians.
- Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
- Statistics of clinical pathology (except for urine color and turbidity): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- Statistics of pathology: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Details on results:

ANALYSES
- Stability analysis: The stability of the test substance in drinking water was demonstrated over a period of 4 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.
- Concentration control analysis of the test substance preparations: 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 94.8 - 102% of the nominal concentrations. These results demonstrate the correctness of the concentrations of Ethylene Urea.
- Food analyses: On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1×105/g food.
- Drinking water analyses: On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.
- Bedding and enrichment analyses: On the basis of the analytical findings the bedding and the enrichment are found to be suitable. Levels given in Lab. Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum tolerable contaminants.

CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study. No abnormal clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
Body weight was significantly decreased in females of test group 3 (2000 ppm) on gestation day 20 (-6.2%) and during lactation days 1 and 4 (up to -9.9%). Body weight change value was significantly reduced in males of test group 3 (2000 ppm) from day 0 to 7 (-38.3%). Body weight change values were significantly reduced during gestation days 14-20 (-24.3%) and during the entire gestation period from day 0-20 in female animals of test group 3 (2000 ppm).

FOOD CONSUMPTION
Food consumption during gestation was significantly decreased in females of test group 3 (2000 ppm) from study days 7-14 and 14-20 (up to -9.5%) and during the entire gestation period from day 0 to 20 (-8.8%). During lactation period the food consumption was significantly decreased (-40.2%) in females of test group 3 (2000 ppm).

WATER CONSUMPTION
Water consumption was significantly reduced in females of test group 3 (2000 ppm) on lactation day 4 (-37.9%).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The intake of the test substance (in mg/kg bw/d) was calculated on the basis of most recent individual body weights in each test group:
Test group 1 Test group 2 Test group 3
(100 ppm) (500 ppm) (2000 ppm)
F0 males (premating) 7.4 36.9 155.0
F0 females (premating) 10.2 47.8 191.0
F0 females (gestation) 11.0 53.1 222.0
F0 females (lactation) 17.0 86.6 234.2

HAEMATOLOGY
No treatment-related, adverse changes among hematological parameters were observed.
In females of test group 3 (2000 ppm) hemoglobin values were lower compared to controls. This was the only altered red blood cell parameter and the decrease was marginal (mean: -6.6%) although below the historical control range (hemoglobin 8.7-9.5 mmol/L). Therefore, this alteration was regarded as treatment-related but not adverse (Müller et al., 2006, ECETOC Technical Report No. 85, 2002).

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.
In females of test group 3 (2000 ppm), creatinine levels were higher compared to controls, but the mean was within the historical control range (creatinine 52.2-62.7 mmol/L, PART III, Supplement) whereas the control mean was below the historical range. Creatinine was the only altered clinical chemistry parameter in this study. Therefore, this change was regarded as incidental and not treatment-related (ECETOC, Technical Report No. 85, 2002).

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually:
- Home cage observations: No test substance-related effects were observed.
- Open field observations: No test substance-related effects were observed.
- Sensorimotor tests/reflexes: No test substance-related effects were observed.
- Quantitative Parameters: The values of landing foot splay test in males of test group 2 (500 ppm) were significantly increased (+27.4%). Due to the lack of dose response relationship this was assessed as being incidental.

Motor activity measurement
Male animals of test group 1, 2 and 3 (100; 500 and 2000 ppm) showed a significantly lower value at interval 1 when compared to control males. Female animals of test group 3 (2000 ppm) showed a significantly lower value in interval 7. There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals. All described findings were assessed as being incidental and not related to treatment.

ORGAN WEIGHTS
When compared to the control group 0 (set to 100%), the mean absolute weight of the thyroid glands was significantly increased in males of test group 3 (2000 ppm). All other mean absolute weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group 0.
When compared to the control group 0 (set to 100%), the mean relative weight of the thyroid glands was significantly increased in males of test group 3 (2000 ppm). All other mean relative weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group 0. The increased thyroid weights in males of test group 3 (2000 ppm) were considered to be treatment-related.

GROSS PATHOLOGY
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The female animals (Nos. 119, 134, 138), which were not pregnant as well as the male mating partners (Nos. 19, 34, 38) did not show relevant gross lesions.

HISTOPATHOLOGY
- Thyroid gland: The number of animals with follicular hypertrophy/ hyperplasia was increased in males and females of test group 3 (2000 ppm). In affected animals the number of small follicles was increased or the follicular epithelium was higher, varying in size from cuboidal cells to columnar cells. The occurrence of hypertrophy/ hyperplasia in animals of test group 3 (2000 ppm) was considered to be treatment-related.
- Axillary lymph nodes and spleen: In the axillary lymph nodes of control males and females, most follicles were primary follicles; these are non-stimulated follicles without germinal centers. In some treated animals, the number of follicles with germinal centers (secondary follicles) was slightly (grade 2) or clearly (grade 4) increased. In the spleen of control male and female animals most follicles had no germinal centers. In some treated males and females the number of follicles with germinal centers was slightly (grade 2) or clearly (grade 4) increased when compared with control animals. The number of males and females with a higher number of secondary follicles (follicles with germinal centers) in the axillary lymph nodes or in the spleen was dose-related increased in all treatment groups. The increase of follicles with germinal centers was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The female animals (Nos. 134, 138), which were not pregnant as well as the male mating partners (Nos. 34, 38) did not show histopathological findings explaining why the animals were not pregnant. From the female animal (No. 119) which was recorded as not pregnant as well as from the male mating partner (No 19) only the axillary lymph node and the spleen were examined histologically, according to the study plan.

Key result

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on decreased body weight/body weight gain, decreased food consumption, and the occurrence of hypertrophy/ hyperplasia in the thyroid glands in males and females of test group 3 (2000 ppm).

Key result

Dose descriptor:

NOAEL

Effect level:

37 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on decreased body weight/body weight gain, decreased food consumption, and the occurrence of hypertrophy/ hyperplasia in the thyroid glands in males and females of test group 3 (2000 ppm).

Key result

Dose descriptor:

NOAEL

Effect level:

57 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on the decreased body weight/body weight gain and decreased food consumption.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Absolute organ weights:

	Male animals
Test group (ppm)	1 (100)	2 (500)	3 (2000)
Thyroid glands	94%	110%	125%**

**p <= 0.01

Relative organ weights:

	Male animals
Test group (ppm)	1 (100)	2 (500)	3 (2000)
Thyroid glands	95%	112%	129%**

** p<=0.01

Thyroid gland - incidence and grading of follicular hypertrophy/ hyperplasia:

Thyroid gland	Male animals				Female animals
Test group (ppm)	0	1 (100)	2 (500)	3 (2000)	0	1 (100)	2 (500)	3 (2000)
Organs examined	10	10	10	10	10	10	10	10
Hypertrophy/ hyperplasia follicular	1		1	10	0	1	1	5
Grade 1	1			1		1		2
Grade 2			1	7			1	3
Grade 3				2

Axillary lymph nodes and spleen - incidence and grading of increase of germinal centers:

Axillary lymph nodes	Male animals				Female animals
Test group (ppm)	0	1 (100)	2 (500)	3 (2000)	0	1 (100)	2 (500)	3 (2000)
Organs examined	10	10	8	10	10	9	10	10
No. germinal center increased		2	3	7		3	8	9
Grade 2		1		4		3	1	1
Grade 4		1	3	3			7	8

Spleen	Male animals				Female animals
Test group (ppm)	0	1 (100)	2 (500)	3 (2000)	0	1 (100)	2 (500)	3 (2000)
Organs examined	10	10	10	10	10	10	10	10
No. germinal center increased	0	3	7	6	0	2	8	9
Grade 2			1	1		2	3	1
Grade 4		3	6	5			5	8

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-imidazolidone

EC Number:

204-436-4

EC Name:

2-imidazolidone

Cas Number:

120-93-4

Molecular formula:

C3H6N2O

IUPAC Name:

2-imidazolidone

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 11-0041

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-12 wks (males/females)
- Weight at study initiation (mean): (P) Males: 316.6 g; Females: 210.6 g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4 (end of lactation).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Type NGM E-022) supplied by Abed® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.
- Diet: Ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum.
- Water: drinking water, ad libitum
- Acclimation period: 1 week

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

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired weight, subsequently released with a magnetic stirrer. The test substance preparations were produced at least twice a week.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: from about 16.00 h until 07.00 - 09.00 h of the following morning, for a maximum of 2 weeks
- 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:

- Analyses of the test substance preparations: The analyses of the test substance preparations were carried out as a separate study at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study at a concentration of 5000 ppm. To verify the stability for the lowest concentration in the current study (100 ppm) a stability analysis was carried out during the study period. Concentration control Analysis of the test substance preparations were performed in samples of all concentrations at the start and towards the end of the administration period.

Duration of treatment / exposure:

Males were exposed for 30 days, females were exposed for 51 days

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 13-14 weeks. In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

500 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 ppm (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: at the request of the sponsor
- Rationale for animal assignment: the animals were distributed according to weight among the individual test groups, separated by sex

Examinations

Parental animals: Observations and examinations:

- Mortality: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.
- Clinical observations: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
- Detailed clinical observations: Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during “handling", fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine and pupil size.
- Water consumption: Generally, water consumption was determined once a week for male and female parental animals, with the following exceptions:
Water consumption was not determined during the mating period (male and female F0 animals), Water consumption of the F0 females with evidence of sperm was determined on GD 7, 14 and 20. And water consumption of F0 females, which gave birth to a litter was determined for PND 4.
Water consumption was not determined in 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) and in males after the premating period.
- Food consumption: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
Food consumption was not determined during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on GD 7, 14 and 20. Food consumption of F0 females, which gave birth to a litter was determined for PND 4. Food consumption was not determined in 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) and in males after the premating period.
- Body weight data: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 1) and on PND 4. Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Intake of test substance: The intake of test substance was calculated from the amount of water consumed and expressed in mg/kg body weight per day
- Functional observational battery: A functional observational battery (FOB) was performed in the first five parental males and females (with litter) per group and the groups were selected at the end of the administration period starting at about 10:00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Motor activity assessment: The motor activity assessment (MA) was carried out in the first five parental males and females (with litter) per group at the end of the administration period. Motor activity was measured on the same day as FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out starting at 14:00 h. On account of the measuring variant "staggered", the starting time varied according to the time needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements. After the transfer of the last animal the room of measurement was darkened. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.
- Male reproduction data: For the males, mating and fertility indices were calculated for F1 litters.
- Female reproduction and delivery data: For the females, mating, fertility and gestation indices were calculated for F1 litters.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight, stages of spermatogenesis

Litter observations:

- Pup number and status at delivery: All pups delivered from the F0 parents 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 pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.
- Pup viability/mortality: 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.
- Pup clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Sex ratio: On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.
- Pup clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Pup body weight data: The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [about two weeks after mating]
- Maternal animals: All surviving animals [about two weeks after parturition]

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

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

HISTOPATHOLOGY
The following tissues were prepared for microscopic examination and weighed: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating gland, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal glands, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

CLINICAL PATHOLOGY: In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Hematology: The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Parameters: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mena corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelt count (PLT), Differnetial blood count and Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).Parameter: Prothrombin time (Hepato Quick's test) (HQT).
- Clinical chemistry: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), y-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (Cl), Inorganic phosphate (INP), Calcium(Ca), Urea (UREA), Creatine (CREA), Glugose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), GLobulins (GLOB), Triglycerides (TRIG), Chlolesterol (CHOL), MAgnesium (MG).
- Urinalysis: The dry chemical reactions on test strips (Combur 10 test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany). pH, Protein, Glucose, ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color and turbidity, Volume.

Postmortem examinations (offspring):

- Pup necropsy observations: All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, viability index: Pairwise comparison of each dose group with the control group using FISHER'S EXACT-test for the hypothesis of equal proportions.
- Number of mating days: Pairwise comparison of the dose group with the control group using the WILCOXON-test (one-sided) with Bonferoni-Holm-Adjustment for the hypothesis of equal medians.
- Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
- Statistics of clinical pathology (except for urine color and turbidity): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- Statistics of pathology: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Reproductive indices:

The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas

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

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

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

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

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

Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
* defined as the number of females with implants in utero

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:

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

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

Post implantation loss (%) = (number of implantations - number of pups delivered/number of implantations) x 100

Offspring viability indices:

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:

Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

ANALYSES
- Stability analysis: The stability of the test substance in drinking water was demonstrated over a period of 4 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.
- Concentration control analysis of the test substance preparations: 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 94.8 - 102% of the nominal concentrations. These results demonstrate the correctness of the concentrations of Ethylene Urea.
- Food analyses: On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1×105/g food.
- Drinking water analyses: On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.
- Bedding and enrichment analyses: On the basis of the analytical findings the bedding and the enrichment are found to be suitable. Levels given in Lab. Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum tolerable contaminants.

CLINICAL EXAMINATIONS AND EXAMINATION OF REPRODUCTIVE PERFORMANCE: Parental clinical examinations and examination of reproductive performance:
- Mortality: No animal died prematurely in the present study.
- Detailed clinical observations: No abnormal clinical signs were observed.
- Clinical observations for females during gestation: One sperm-positive F0 female of test group 1 (No. 119) and two sperm-positive F0 female of the test group 3 (Nos. 134 and 138) did not deliver any F1 pups. No other clinical signs were observed.
- Clinical observations for females during lactation: One female animal of test group 3 showed complete litter loss. One female animal of test group 1 showed insufficient maternal care and did not nurse the pups properly during lactation period.
- Water consumption: Water consumption was significantly reduced in females of test group 3 (2000 ppm) on lactation day 4 (-37.9%).
- Food consumption: Food consumption during gestation was significantly decreased in females of test group 3 (2000 ppm) from study days 7-14 and 14-20 (up to -9.5%) and during the entire gestation period from day 0 to 20 (-8.8%). During lactation period the food consumption was significantly decreased (-40.2%) in females of test group 3 (2000 ppm).
- Body weight data: Body weight was significantly decreased in females of test group 3 (2000 ppm) on gestation day 20 (-6.2%) and during lactation days 1 and 4 (up to -9.9%). Body weight change value was significantly reduced in males of test group 3 (2000 ppm) from day 0 to 7 (-38.3%). Body weight change values were significantly reduced during gestation days 14-20 (-24.3%) and during the entire gestation period from day 0-20 in female animals of test group 3 (2000 ppm)

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The intake of the test substance (in mg/kg bw/d) was calculated on the basis of most recent individual body weights in each test group:
Test group 1 Test group 2 Test group 3
(100 ppm) (500 ppm) (2000 ppm)
F0 males (premating) 7.4 36.9 155.0
F0 females (premating) 10.2 47.8 191.0
F0 females (gestation) 11.0 53.1 222.0
F0 females (lactation) 17.0 86.6 234.2
- Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually: - Home cage observations: No test substance-related effects were observed, - Open field observations: No test substance-related effects were observed. - Sensorimotor tests/reflexes: No test substance-related effects were observed. - Quantitative Parameters: The values of landing foot splay test in males of test group 2 (500 ppm) were significantly increased (+27.4%). Due to the lack of dose response relationship this was assessed as being incidental.
- Motor activity measurement: Male animals of test group 1, 2 and 3 (100; 500 and 2000 ppm) showed a significantly lower value at interval 1 when compared to control males. Female animals of test group 3 (2000 ppm) showed a significantly lower value in interval 7. There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals. All described findings were assessed as being incidental and not related to treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Males
For 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.
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter.
One male of test group 1 (No. 19 mated with female No. 119) and two males of test group 3 (Nos.34 and 38 mated with female Nos. 134 and 138) did not generate F1 pups.
-The male fertility index was between the range of 80% and 100%.
Females
-The female mating index calculated after the mating period for F1 litter was 100% for all test groups The mean duration until sperm was detected (GD 0) was 2.3, 3.4, 3.3 and 2.2 days in test groups 0-3.
All sperm positive rats delivered pups with the exception of female Nos. 119 (test group 2), 134 and 138 (test group 3), which were mated with male Nos. 19, 34 and 38 did not become pregnant. The female fertility index was 80% in the high dose group, 100% in the mid dose and control group and 90% in the low dose group. Female animals Nos. 119, 134 and 138 which delivered no pups, showed no implantation sites.

-The gestation index was 100% in all test groups.
-The rate of liveborn pups was between 99.2% (test group 0), 99.0 (test group 1), 99.1 (test group 2) and 97.4% (test group 3). One stillborn pup was seen in test group 0-2 (0 ppm, 100 ppm and 500 ppm) and two stillborn pups were seen in test group 3 (2000 ppm). Thereby, the live birth index was in the range of the rat strain used.
-The postimplantation loss was between 18.12% (test group 0), 5.61% (test group 1), 5.65% (test group 2) and 9.52% (test group 3). The value in test group 0 was unusually high, but all other values were inside the historical control data.

CLINICAL PATHOLOGY
- Hematology: No treatment-related, adverse changes among hematological parameters were observed. In females of test group 3 (2000 ppm) hemoglobin values were lower compared to controls. This was the only altered red blood cell parameter and the decrease was marginal (mean: -6.6%) although below the historical control range (hemoglobin 8.7-9.5 mmol/L). Therefore, this alteration was regarded as treatment-related but not adverse (Müller et al., 2006, ECETOC Technical Report No. 85, 2002).
- Clinical chemistry: No treatment-related changes among clinical chemistry parameters were observed. In females of test group 3 (2000 ppm), creatinine levels were higher compared to controls, but the mean was within the historical control range (creatinine 52.2-62.7 mmol/L) whereas the control mean was below the historical range. Creatinine was the only altered clinical chemistry parameter in this study. Therefore, this change was regarded as incidental and not treatment-related (ECETOC, Technical Report No. 85, 2002)
- Urinalyses: No treatment-related changes among urinalysis parameters were observed.

PATHOLOGY
- ORGAN WEIGHTS (PARENTAL ANIMALS)
When compared to the control group 0 (set to 100%), the mean absolute weight of the thyroid glands was significantly increased in males of test group 3. All other mean absolute weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group 0.
When compared to the control group 0 (set to 100%), the mean relative weight of the thyroid glands was significantly increased in males of test group 3. All other mean relative weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group 0. The increased thyroid weights in males of test group 3 (2000 ppm) were considered to be treatment-related.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The female animals (Nos. 119, 134, 138), which were not pregnant as well as the male mating partners (Nos. 19, 34, 38) did not show relevant gross lesions.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The number of animals with follicular hypertrophy/ hyperplasia was increased in males and females of test group 3 (2000 ppm). In affected animals the number of small follicles was increased or the follicular epithelium was higher, varying in size from cuboidal cells to columnar cells. The occurrence of hypertrophy/ hyperplasia in animals of test group 3 (2000 ppm) was considered to be treatment-related.
- Axillary lymph nodes and spleen: In the axillary lymph nodes of control males and females, most follicles were primary follicles; these are non-stimulated follicles without germinal centers. In some treated animals, the number of follicles with germinal centers (secondary follicles) was slightly (grade 2) or clearly (grade 4) increased. In the spleen of control male and female animals most follicles had no germinal centers. In some treated males and females the number of follicles with germinal centers was slightly (grade 2) or clearly (grade 4) increased when compared with control animals. The number of males and females with a higher number of secondary follicles (follicles with germinal centers) in the axillary lymph nodes or in the spleen was dose-related increased in all treatment groups. The increase of follicles with germinal centers was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Fertility: The female animals (Nos. 134, 138), which were not pregnant as well as the male mating partners (Nos. 34, 38) did not show histopathological findings explaining why the animals were not pregnant. From the female animal (No. 119) which was recorded as not pregnant as well as from the male mating partner (Nos. 19) only the axillary lymph node and the spleen were examined histologically, according to the study plan.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

2

Key result

Dose descriptor:

NOAEL

Effect level:

155 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

3

Key result

Dose descriptor:

NOAEL

Effect level:

214 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

Details on results (F1)

- Pup viability/mortality: The viability index indicating pup mortality during lactation (PND 0 - 4) was between 98.5% (test group 0), 100% (test group 1), 98.4% (test group 2) and 81.6% (test group 3). For test group 0-2, these values were in the normal range of biological variation inherent in the strain of rats used for this study. For test group 3, a decreased pup viability was noted, which was also outside the historical control range. Although 7 to the 11 dead offsprings in this group came from only one litter (No. 135), a relationship to the treatment cannot be excluded.
- Pup number and status at delivery: The mean number of delivered F1 pups per dam was evenly distributed about the groups. The mean number of delivered F1 pups was 12.2 (test group 0), 11.2 (test group 1), 11.7 (test group 2) and 9.5 (test group 3). The single stillborn pup each in test group 0, 1 and 2 and two stillborn pups in test group 3 were incidental and in the normal range of biological variation inherent in the strain of rats used for this study.
- Sex ratio: The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
- Body weight: Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
In test group 1 (100 ppm) three male runts and one female runt were seen (Animal No. 111). In test group 2 (500 ppm) one male runt was seen in animal nos. 123 and 127. In test group 3 (2000 ppm) four male and three female runts were seen in animal no. 132 and one male runt in animal no. 139 was seen. In animal no. 140 four male runts were seen.
- Pup clinical observations: The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
- Gross pathology: One stillborn pup of test group 0 (0 ppm) showed post mortem autolysis. Four pups of Animal No. 135 (test group 3; 2000 ppm) showed an empty stomach. One pup of Animal No. 131 showed a dilated renal pelvis and one pup of Animal No. 139 showed a discolored liver. These findings were assessed as being spontaneous in nature and without biological relevance.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on decreased pup viability.

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

37 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on decreased pup viability.

3

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

57 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on decreased pup viability.

Overall reproductive toxicity

Reproductive effects observed:

not specified