2-imidazolidone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined repeated dose toxicity study with the reproduction/toxicity screening test (OECD 422), no signs of toxicity regarding fertility and reproductive performance were observed in male or female parental animals of all test groups (100, 500 and 2000 ppm in drinking water).

Supportingly, no adverse effects regarding reproductive parameters were observed in an OECD Guideline 408 study with additional examinations of male and female reproductive parameters (oestrous cycle, sperm parameters, and reproductive and other certain organs and tissues).

Link to relevant study records

Reference

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

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

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 (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

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".

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

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

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.

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

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

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

Key result

Critical effects observed:

no

- 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.

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.

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.

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.

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

155 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP compliant OECD guideline 422 study, 2-imidazolidone was administered orally via drinking water to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 ppm (test group 0), 100 ppm (test group 1), 500 ppm (test group 2) and 2000 ppm (test group 3) (BASF SE, 2013).

The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1-week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females.

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-2 (100 and 500 ppm) during the entire study.

The decreased body weight changes in males during the premating period from day 0 to 7 (-38.3%) and the decreased food consumption during the entire gestation period and the decreased body weight during gestation days 1 and 4 (up to -9.9%) in the test group 3 (2000 ppm) were considered to be adverse and toxicologically relevant effects.

Regarding clinical pathology, no treatment-related effects were observed up to a dose of the compound of 2000 ppm. Regarding pathology, target organs were the thyroid glands, the axillary lymph nodes and the spleen. In the thyroid glands, a diffuse hypertrophy/ hyperplasia of follicular epithelium was observed in all males (minimal to moderate) and in five out of 10 females (minimal or slight) of test group 3 (2000 ppm). Hypertrophy/ hyperplasia of follicular epithelium was regarded to be responsible for the recorded weight changes in males of test group 3. The occurrence of hypertrophy/ hyperplasia in males and females of test group 3 (2000 ppm) was considered to be treatment-related and adverse.

In the axillary lymph nodes of control males and females most follicles represented primary follicles, only few secondary follicles (follicles with germinal centers) were noted. After treatment with the test substance, some males and females showed a higher number of secondary follicles with germinal centers. A comparable effect was observed in the spleen. The number of males and females with a higher number of secondary follicles in the axillary lymph nodes or in the spleen was dose-related increased in all treatment groups. There were no weight changes and no further histopathological findings in lymphatic organs. Furthermore, no changed parameters in the clinical pathology were noted. The increase of follicles with germinal centers was considered to be treatment-related.

An increase of germinal centers can be caused by immunostimulation. According to the literature only a few compounds have immunostimulating or immunoadjuvant properties. An example of an agent with immunopotentiating properties is Hexachlorobenzene (HCB). In rats, prominent changes following dietary exposure include elevated IgM levels and an increase in the weights of the spleen and lymph nodes (Kuper, F. et al, 2000). Histopathologically, the spleen shows increased extramedullary hemopoiesis and hyperplasia of B lymphocytes in the marginal zone and follicles. Lymph nodes and Peyer’s patches show an increase in proportions of high endothelial venules (HEV), indicative of activation. Apart from the increase in germinal centers, these findings were not observed in the present study. Immunostimulation can also be seen in combination with inflammation; however, there were no signs of inflammation in any investigated organ in the present study. The cause of the increase of germinal centers is unclear. However, the development of germinal centers is a normal reaction after antigen stimulation and the increase of germinal centers was the only finding in the lymphatic organs. Therefore, the increase of germinal centers was regarded as non-adverse.

All other findings recorded were considered to be incidental in nature and not related to treatment.

Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups (100, 500 and 2000 ppm) during the entire study.

The NOAEL for reproductive performance and fertility was 2000 ppm (155 mg/kg bw/day in parental males and 214 mg/kg bw/day in parental females) for the F0 parental rats.

Supportingly, no adverse effects regarding reproductive parameters were observedin an OECD Guideline 408 study with additional examinations of male and female reproductive parameters (oestrous cycle, sperm parameters, and reproductive and other certain organs and tissues) (BASF SE, 2018):

- Estrous cycle: No test substance-related effects on estrous cycle length and the number of cycles were obtained.

- Sperm parameters: Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

- There were no gross lesions or treatment-related histopathological findings in reproductive organs and tissues.

 

Effects on developmental toxicity

Description of key information

In a combined repeated dose toxicity study with the reproduction/toxicity screening test (OECD 422), signs of toxicity were observed in a decreased pup viability index in test group 3 (2000 ppm) and in one litter loss, wherefore a relationship to the treatment cannot be excluded. The NOAEL for developmental toxicity in the F1 progeny was found to be 500 ppm (37 mg/kg bw /day for male rats and 57 mg/kg bw/day for female rats) based on the decreased pup viability which was likely to be subsequent to insufficient maternal care.

In a prenatal developmental toxicity study (OECD 414), the oral administration of 2-Imidazolidone to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 1000 mg/kg bw/d caused signs of maternal toxicity, such as a reduction in food consumption and a decrease in (corrected) body weight (change/gain). The NOAEL for maternal toxicity was established at the mid dose of 200 mg/kg bw/d. The NOAEL for prenatal developmental toxicity was the highest tested dose of 1000 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-01-2017 - 07-05-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

The analyses of the test item (= test substance) were carried out at Competence Center Ana- lytics, BASF SE, 67056 Ludwigshafen, Germany.

Name of test substance: 2-Imidazolidone
Test substance No.: 08/0054-5
Batch identification: 16960025
CAS No.: 120-93-4
Purity: 89.2 corr. area-% (GC)
(Report, Study code: 16L00102)
Content: 89.7 g/100 g (1H-NMR) (Report, Study code: 16L00102)
Identity: Confirmed (Report, Study code: 16L00102)
Homogeneity: Given
Storage stability: Expiry date: 28 Feb 2017
Storage conditions: Room temperature

The stability of the test substance under storage con- ditions over the test period was guaranteed by the sponsor, and the sponsor holds this
responsibility. The test facility is organizationally in- dependent from the BASF SE sponsor division.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species and strain
Time-mated Wistar rats (Crl:WI[Han]) were supplied by Charles River Laboratories, Research Models and Services, Germany GmbH, at an age of about 10-12 weeks. Only animals free from clinical signs of disease were used for the investigations.

Animal identification
The animals were paired by the breeder and supplied on GD 0 (= detection of vaginal plug/sperm). After arrival of each cohort, the assignment of the animals to the different test groups was carried out by withdrawing them from the transport boxes and placing them into the cages at random. Each cohort was evenly distributed among the dose groups. After ran- domization the rats were identified consecutively and uniquely by ear tattoo.

Reason for species selection
The Crl:WI(Han) strain was selected since extensive historical control data is available from the test facility for Wistar rats. This specific strain has been proven to be sensitive to sub- stances with a teratogenic potential.

HOUSING AND DIET
During the study period, the rats were housed individually in Polycarbonate cages type III sup- plied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Ger- many (floor area about 800 cm²). Individual housing is preferred over group housing as the close individual monitoring of food and water consumption as well as of clinical signs of toxicity in pregnant animals is crucial during this period of increased sensitivity. In addition, the control for signs of abortion or fetal loss can only be done in a reliable fashion with single-housed animals.
Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data).
For enrichment, wooden gnawing blocks were offered (Typ Lignocel® block large, supplied by
J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany).
The animals were accommodated in fully air-conditioned rooms in which central air condition- ing maintained a range of temperature of 20-24°C and a range of relative humidity of 30-70%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study.
The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h).

Before the study started, the animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection). The walls and the floor were cleaned once a week with water containing an appropriate disinfectant.
The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bot- tles) were available ad libitum.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Diet preparation
The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The prep- arations were kept at room temperature.

For the test substance preparations, the specific amount of test substance was weighed, topped up with drinking water in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.

Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.


Test group Dose Concentration Volume administered
[mg/kg bw/d] [g/100 mL] [mL/kg bw/d]
0 0 0 10
1 40 0.40 10
2 200 2.00 10
3 1000 10.00 10

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations concerning the stability of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
The concentration control analyses from samples of the current study were carried out as a separate study at the external test facility Pharmacelsus GmbH, Science Park 2, 66123 Saar- brücken, Germany, under the responsibility of the study director of this test facility (BASF Pro- ject No. 02Y0054/08X094). The study was carried out in compliance with the Principles of Good Laboratory Practice.
Analytical verifications of the stability of the test substance in drinking water over a period of 4 days at room temperature were conducted prior to the start of the study in a similar batch (Project No.: 01Y0054/08Y010).
Samples of the test substance preparations were sent to the analytical laboratory at the begin- ning of administration for verification of the concentrations.
Given that the test substance was completely miscible with drinking water, solutions were con- sidered homogenous without further analysis.


The results of the analysis of the test substance preparations confirmed the correctness of the prepared concentrations. The measured concentrations of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= de- tection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The ani- mals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).

Duration of treatment / exposure:

The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning.

Frequency of treatment:

once daily

Duration of test:

14 days

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
Doses were selected based on a preliminary 14 day range finding study. In this study some minor effects were seen in both dose groups tested:

Test group 2 (1000 mg/kg bw/d):
" Increased (not statistically significant) weight of thyroid glands (absolute: +16%, relative: +20%)
* Minimal (grade 1) to moderate (grade 3) hypertrophy/hyperplasia of the thyroid glands in 7/7 animals

Test group 1 (300 mg/kg bw/d):
* Increased (not statistically significant) weight of thyroid glands (absolute: +13%, relative: +15%)
* Minimal (grade 1) to slight (grade 2) hypertrophy/hyperplasia of the thyroid glands in 6/7 animals

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-
15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
On GD20 all surviving dams were sacrificed by decapitation under isoflurane anesthesia in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution:
1. All gross lesions
2. Thyroid gland

No further examinations or procedures were performed in the study.


OTHER:
A check for mortality was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

Clinical Pathology
In the morning blood was taken from the retro-bulbar venous plexus from non-fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subse- quent analysis of blood and serum samples were carried out in a randomized sequence.

Hormones
Serum samples were frozen at -80 degrees Celsius for storage. Measurement of T3, T4 and TSH has been planned if there was an indication for an effect on pituitary-thyroid axis. Sam- ples will not be stored longer than one year after finalization of the report. So far, no investigations have been conducted.

Ovaries and uterine content:

After the dams had been sacrificed, they were necropsied and assessed for gross pathology. The uteri and the ovaries were removed and the following data were recorded:
-Weight of the unopened uterus
-Number of corpora lutea
-Number and distribution of implantation sites classified as:
•Live fetuses
•Dead implantations:

a)Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b)Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c)Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters (except of gross pathology) were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
These data were used to calculate conception rate and pre- and postimplantation losses.

Fetal examinations:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Examinations of the fetuses after dissection from the uterus
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were exam- ined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia.
Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded.
Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus).
After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Statistics:

Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means
Female mortality, females pregnant at terminal sacrifice, number of lit- ters with fetal findings: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one- sided) for the hypothesis of equal proportions
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Indices:

The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals x 100)/number of fertilized animals

The preimplantation loss (in %) for each individual pregnant animal which underwent sched- uled sacrifice was calculated according to the following formula:
((number of corpora lutea – number of implantations) x 100)/number of corpora lutea

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of implantations – number of live fetuses)x 100)/number of implantations


Evaluation criteria for assessing the fetuses
Classification and assessment of fetal findings is a matter of ongoing discussion (see e.g. BEL- TRAME and GIAVINI, CHAHOUD, SOLECKI). Despite considerable efforts to harmonize the nomenclature used to describe observations of fetal morphology, the terms still vary consider- ably between laboratories, investigators and textbooks in the fields of teratology and develop- mental toxicity.
In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI:

Malformation
A permanent structural change that is likely to adversely affect the survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.
All fetal findings were listed in tables according to these classifications.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 40, 200 or 1000 mg/kg bw/d during the entire study period.
However, one high-dose dam (No. 95) had a vaginal hemorrhage after treatment (<2 h) on GD 17. During cesarean section, for this female only one viable fetus, but 10 early resorptions and one late resorption (out of 12 implantation sites) were recorded. Hence, the post-implan- tation loss of this dam was 91.7% whereas for the other test group 3 dams, the mean post- implantation loss, number of early and late resorptions were comparable to the control. Furthermore, historical control data of the last 5 years showed that 4 control dams of 4 different studies had a post-implantation loss between 90 and 100%. Therefore, the above mentioned finding in one individual dam was not assessed as treatment-related and adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 40, 200 or 1000 mg/kg bw/d).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight data: The mean body weights and the average body weight gain of the low- and mid-dose dams (40 and 200 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
Body weight change of the high-dose dams was decreased compared to control (around -10%, without statistical significance) during GD 6-19. Together with the reduction in food consumption, the decrease in body weight (change) in the high-dose dams was assessed as treatment-related and adverse.

Corrected (net) body weight gain: The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of test group 3 (1000 mg/kg bw/d) was statistically signifi- cantly reduced in comparison to the control group (-13%). This was assessed as treatment- related and adverse.
The corrected body weight gain of test groups 1 and 2 (40 and 200 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group.
Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the dams in test group 3 (1000 mg/kg bw/d) was statistically significantly reduced at the end of gestation during GD 19-20 (-15 % in comparison to the concurrent control). If calculated for the entire treatment (GD 6-19) period, the high-dose dams consumed about 4% less food than the controls. This was assessed as treatment-related.
For the mid-dose dams (200 mg/kg bw/d), the mean food consumption was statistically significantly reduced at the end of gestation during GD 19-20 (-12 % in comparison to the concurrent control). However, the reduction was assessed as marginal since it occurred in a short time period and it was the only changed parameter. Therefore, it was not assessed as treatment-related and adverse.
The mean food consumption of the dams in test group 1 (40 mg/kg bw/d) was comparable to the concurrent control group throughout the entire study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Uterus weight: The mean gravid uterus weights of the animals of test groups 1-3 (40, 200 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
The mean placental weights of the low-, mid- and high-dose groups were comparable to the corresponding control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Focal constriction in the liver of one animal of the low-dose group (40 mg/kg bw/d). The single macroscopic finding occurred individually. It was considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

see "details on maternal toxic effects" below

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

see "details on maternal toxic effects" below

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

see "details on maternal toxic effects" below

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

see "details on maternal toxic effects" below

Dead fetuses:

no effects observed

Description (incidence and severity):

see "details on maternal toxic effects" below

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

see "details on maternal toxic effects" below

Details on maternal toxic effects:

Reproduction data: The conception rate was 92% in the control group (0 mg/kg bw/d), 96% in the mid- and high- dose groups (200 and 1000 mg/kg bw/d) and 100% in the low-dose group (40 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study.
There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 40, 200 and 1000 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
However, one high-dose dam (No. 95) had a vaginal hemorrhage after treatment (<2 h) on GD 17. During cesarean section, for this female only one viable fetus, but 10 early resorptions and one late resorption (out of 12 implantation sites) were recorded. Hence, the post-implantation loss of this dam was 91.7% whereas for the other test group 3 dams, the mean post-implantation loss, number of early and late resorptions were comparable to the control. Furthermore, historical control data of the last 5 years showed that 4 control dams of 4 different studies had a post-implantation loss between 90 and 100%. Therefore, the above mentioned finding in one individual dam was not assessed as treatment-related and adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat. (total fraction)

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (40, 200 and 1000 mg/kg bw/d) was comparable to the control fetuses.

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were seen in one fetus of a low-dose litter (40 mg/kg bw/d) and in one fetus of a high-dose litter (1000 mg/kg bw/d). All external malfor- mations were associated with skeletal malformations. Fetuses of the mid-dose showed no malformations.
For the one affected fetus of test group 1 multiple external malformations were recorded. The finding multiple external malformations was not related to dose and was within the range of the historical control data (HCD, affected fetuses per litter: range 0.0 - 0.4%).
The finding gastroschisis in the high-dose fetus was well within the range of the historical con- trol data (HCD, affected fetuses per litter: range 0.0 - 0.7%).
Therefore, both malformations were not assessed as treatment-related and adverse.
The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was comparable to the historical control data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in two fetuses each in test groups 1 and 3 (40 and 1000 mg/kg bw/d). One low-dose fetus (No. 46-05) had multiple skeletal malformations, affecting the skull, vertebral column and ribs, associated with multiple external malformations. Furthermore, for one high-dose fetus (No. 86-08), three skeletal malformations associated with an external malformation (gastroschisis) were recorded. The incidences of the total skeletal malformations were not dose-dependent.

All findings, except of fused thoracic arch and split scapula, can be found in the historical con- trol data at comparable or higher incidences. The findings fused thoracic arch and split scapula were single events in individual fetuses. There was no relation to dose. Therefore, they were not considered to be treatment-related.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.

For a better overview, all skeletal variations with statistically significant differences between the control and any treated group were compiled in the table below. All incidences were expressed on a fetus per litter basis and any statistically significant differences which were outside historical control ranges were marked with*.


Finding Test group 0 Test group 1 Test group 2 Test group 3 HCD
0 mg/kg bw/d 40 mg/kg bw/d 200 mg/kg bw/d 1000 mg/kg bw/d Mean % (range)
Incomplete ossification
of nasal; unchanged
cartilage 0.0 0.0 4.1* 1.3 1.1 (0.0 – 4.4)
Incomplete ossification
of sacral arch;
cartilage present 0.0 5.7 8.2* 5.5* 1.4 (0.0 – 8.1)
Incomplete ossification
of sternebra;
unchanged cartilage 75.7 67.7 74.1 88.9* 86.4 (69.3 – 95.5)

The finding ‘incomplete ossification of nasal; unchanged cartilage’ was recorded in test groups 2 and 3, attaining statistical significance in test group 2. However, all values were within the historical control range (see table above). Therefore, the finding was not assessed as treatment-related.
The finding ‘incomplete ossification of sacral arch; cartilage present’ was statistically signifi- cantly increased in test groups 2 and 3. For test group 3, the mean value was within range of the historical control data whereas, for test group 2, it was at the upper border of the range of the HCD. However, the increase was not dose-related and, therefore, it was not assessed as treatment-related.
The finding ‘incomplete ossification of sternebra; unchanged cartilage’ attained statistical significance in test group 3. However, the finding was within the range of the historical control data. Therefore, this finding was not assessed as treatment-related.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum. The overall incidences of skeletal unclassified cartilage observations in the substance-treated groups did not differ significantly from the concurrent control group.

The incidence of ‘notched cartilage between basisphenoid and basioccipital’ was statistically significantly increased in test groups 1 and 3 (affected fetuses/litter, mean [%]: 0.9/6.4*/4.8/6.3* [*=p≤0.05]). Since this finding showed no dose-dependency and all incidences were well within the historical control range (affected fetuses per litter: mean 2.7%, [0.0 - 10.0%]), it was not assessed as treatment-related and adverse.

The incidence of ‘branched rib cartilage’ was statistically significantly increased in test groups 1-3 (affected fetuses/litter, mean [%]: 1.3/4.5*/6.0*/6.2* [*=p≤0.05]). However, the values of all test groups were clearly within the historical control range (affected fetuses per litter: mean 2.1% [0.0 - 7.0%]) and there was no relation to dosing. Thus, this finding was not assessed as treatment-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus of test group 1 (40 mg/kg bw/d) had a soft tissue malformation. The finding diaphragmatic hernia was not related to dose and within the range of the historical control data (HCD, affected fetuses/litter: range of 0.0 – 0.9%). Therefore, it is not assessed as treatment-related.
The overall incidences of soft tissue malformations were comparable to those found in the historical control data

Three soft tissue variations were detected, i.e. short innominate, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly nor dose-de- pendently increased in the treated groups. All of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related and adverse.

No soft tissue unclassified observations were recorded.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

One external variation, i.e. limb hyperextension, was recorded in each one fetus of test groups 2 and 3 (200 and 1000 mg/kg bw/d). The incidence of this single finding was not statistically significantly different from control. It was not related to dose and can be found in the historical control data at comparable or higher incidences (HCD of fetal incidence: 0.05%, [0.0 – 0.8] and HCD of affected fetuses per litter: mean 0.1%, [0.0 - 0.7]). Thus, it is not considered as treatment-related and adverse.

No external unclassified observations were recorded.

Details on embryotoxic / teratogenic effects:

There were noted external, soft tissue and skeletal malformations in test groups 1 and 3 (40 and 1000 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose.

Three fetuses were multiple-malformed. Male low-dose fetus No. 43-04 (40 mg/kg bw/d) had a shortened scapula and shortened humerus. Female low-dose fetus No. 46-05 (40 mg/kg bw/d) had multiple external malformations affecting head and trunk, in conjunction with multiple skeletal malformations affecting skull, vertebral column and ribs. High-dose female fetus No. 86-08 (1000 mg/kg bw/d) showed gastroschisis, shortened scapula, fused thoracic arch as well as cleft sternum and had a distinctly lower fetal body weight compared to the group mean value (2.4 g vs. 3.6 g). Further malformations, i.e. diaphragmatic hernia and split scapula were observed in individual fetuses, unrelated to the dose.

All these findings were single cases, some of them can be found in the historical control data. Specifically, shortened scapula and humerus are common findings in the respective rat strain without any consequence on postnatal development. No ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study. There is no evidence for any association of these scattered findings with the treatment.

One external variation, three soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. None of the total incidences showed a relation to dose. The majority of individual variations were equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency.

No unclassified external and unclassified soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3 (0, 40, 200 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment.

Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to the highest dose tested (1000 mg/kg bw/d).

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat. (dissolved fraction)

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

Individual fetal external malformations:

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (40 mg/kg bw/d)	46-05 F	multiple external malformations
2 (200 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	86-08 F	gastroschisis

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

 

Total external malformations:

		Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 216	25 263	24 243	24 228
Fetal incidence	N (%)	0.0	1 (0.4)	0.0	1 (0.4)
Litter incidence	N (%)	0.0	1 (4.0)	0.0	1 (4.2)
Affected fetuses/litter	Mean%	0.0	0.4	0.0	0.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Total external variations:

		Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 216	25 263	24 243	24 228
Fetal incidence	N (%)	0.0	0.0	1 (0.4)	1 (0.4)
Litter incidence	N (%)	0.0	0.0	1 (4.2)	1 (4.2)
Affected fetuses/litter	Mean%	0.0	0.0	4.2	0.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Individual fetal soft tissue malformations:

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (40 mg/kg bw/d)	41-02 F	diaphragmatic hernia
2 (200 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	none

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

 

Total soft tissue malformations:

		Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 102	25 126	23 117	23 107
Fetal incidence	N (%)	0.0	1 (0.8)	0.0	0.0
Litter incidence	N (%)	0.0	1 (4.0)	0.0	0.0
Affected fetuses/litter	Mean%	0.0	0.7	0.0	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Total soft tissue variations:

		Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 102	25 126	23 117	23 107
Fetal incidence	N (%)	3 (2.9)	3 (2.4)	6 (5.1)	5 (4.7)
Litter incidence	N (%)	3 (14)	3 (12)	4 (17)	4 (17)
Affected fetuses/litter	Mean%	2.8	2.1	7.7	3.8

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Individual fetal skeletal malformations:

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (40 mg/kg bw/d)	43-04 M	shortened scapula, shortened humerus
	46-05 F	multiple skeletal malformations
2 (200 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	86-08 F	shortened scapula, fused thoracic arch, cleft sternum
	96-01 M	split scapula

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

 

Total skeletal malformations:

		Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 114	25 137	24 126	24 121
Fetal incidence	N (%)	0.0	2 (1.5)	0.0	2 (1.7)
Litter incidence	N (%)	0.0	2 (8.0)	0.0	2 (8.3)
Affected fetuses/litter	Mean%	0.0	1.7	0.0	1.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Total fetal skeletal variations:

		Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 114	25 137	24 126	24 121
Fetal incidence	N (%)	111 (97)	131 (96)	121 (96)	120 (99)
Litter incidence	N (%)	22 (100)	25 (100)	24 (100)	24 (100)
Affected fetuses/litter	Mean%	97.8	96.3	96.5	99.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter):

Finding	Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of nasal; unchanged cartilage	0.0	0.0	4.1*	1.3	1.1 (0.0 – 4.4)
Incomplete ossification of sacral arch; cartilage present	0.0	5.7	8.2*	5.5*	1.4 (0.0 – 8.1)
Incomplete ossification of sternebra; unchanged cartilage	75.7	67.7	74.1	88.9*	86.4 (69.3 – 95.5)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p<=0.05 (Wilcoxon-test [one-sided]) ** = p<0.01 (Wilcoxon-test [one-sided])

 

Total unclassified cartilage observations:

		Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 114	25 137	24 126	24 121
Fetal incidence	N (%)	81 (71)	107 (78)	100 (79)	97 (80)
Litter incidence	N (%)	22 (100)	25 (100)	24 (100)	24 (100)
Affected fetuses/litter	Mean%	71.1	78.4	79.4	79.8

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Total fetal malformations:

		Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 216	25 263	24 243	24 228
Fetal incidence	N (%)	0.0	3 (1.1)	0.0	2 (0.9)
Litter incidence	N (%)	0.0	3 (12)	0.0	2 (8.3)
Affected fetuses/litter	Mean%	0.0	1.2	0.0	0.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Total fetal variations:

		Test group 0 0 mg/kg bw/d	Test group 1 40 mg/kg bw/d	Test group 2 200 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 216	25 263	24 243	24 228
Fetal incidence	N (%)	114 (53)	134 (51)	127 (52)	125 (55)
Litter incidence	N (%)	22 (100)	25 (100)	24 (100)	24 (100)
Affected fetuses/litter	Mean%	53.0	51.0	55.3	56.6

mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of 2-Imidazolidone to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 1000 mg/kg bw/d caused signs of maternal toxicity, such as a reduction in food consumption and a decrease in (corrected) body weight (change/gain).

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid dose of 200 mg/kg bw/d.
The NOAEL for prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP compliant OECD guideline 422 study, 2 -imidazolidone was administered orally via drinking water to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 ppm (test group 0), 100 ppm (test group 1), 500 ppm (test group 2) and 2000 ppm (test group 3) (BASF SE, 2013).

The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females.

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-2 (100 and 500 ppm) during the entire study. The decreased body weight changes in males during the premating period from day 0 to 7 (-38.3%) and the decreased food consumption during the entire gestation period and the decreased body weight during gestation days 1 and 4 (up to -9.9%) in the test group 3 (2000 ppm) were considered to be adverse and toxicologically relevant effects. 

Regarding clinical pathology, no treatment-related effects were observed up to a dose of the compound of 2000 ppm. Regarding pathology, target organs were the thyroid glands, the axillary lymph nodes and the spleen.

In the thyroid glands, a diffuse hypertrophy/ hyperplasia of follicular epithelium was observed in all males (minimal to moderate) and in five out of 10 females (minimal or slight) of test group 3 (2000 ppm). Hypertrophy/ hyperplasia of follicular epithelium was regarded to be responsible for the recorded weight changes in males of test group 3. The occurrence of hypertrophy/ hyperplasia in males and females of test group 3 (2000 ppm) was considered to be treatment-related and adverse.

In the axillary lymph nodes of control males and females most follicles represented primary follicles, only few secondary follicles (follicles with germinal centers) were noted. After treatment with the test substance, some males and females showed a higher number of secondary follicles with germinal centers. A comparable effect was observed in the spleen. The number of males and females with a higher number of secondary follicles in the axillary lymph nodes or in the spleen was dose-related increased in all treatment groups. There were no weight changes and no further histopathological findings in lymphatic organs. Furthermore, no changed parameters in the clinical pathology were noted. The increase of follicles with germinal centers was considered to be treatment-related.

An increase of germinal centers can be caused by immunostimulation. According to the literature only a few compounds have immunostimulating or immunoadjuvant properties. An example of an agent with immunopotentiating properties is Hexachlorobenzene (HCB). In rats, prominent changes following dietary exposure include elevated IgM levels and an increase in the weights of the spleen and lymph nodes (Kuper, F. et al, 2000). Histopathologically, the spleen shows increased extramedullary hemopoiesis and hyperplasia of B lymphocytes in the marginal zone and follicles. Lymph nodes and Peyer’s patches show an increase in proportions of high endothelial venules (HEV), indicative of activation. Apart from the increase in germinal centers, these findings were not observed in the present study. Immunostimulation can also be seen in combination with inflammation; however there were no signs of inflammation in any investigated organ in the present study. The cause of the increase of germinal centers is unclear. However, the development of germinal centers is a normal reaction after antigen stimulation and the increase of germinal centers was the only finding in the lymphatic organs. Therefore, the increase of germinal centers was regarded as non-adverse.

All other findings recorded were considered to be incidental in nature and not related to treatment.

Regarding developmental toxicity, signs of toxicity were observed in the decreased pup viability index in test group 3 (2000 ppm) and in one litter loss, wherefore a relationship to the treatment cannot be excluded. However, at necropsy four pups of the dam with complete litter loss (animal No. 135, test group 3; 2000 ppm) had an empty stomach indicating that this pup mortality was likely to be subsequent to insufficient maternal care.

The NOAEL for developmental toxicity in the F1 progeny was found to be 500 ppm (37 mg/kg bw /day in parental males and 57 mg/kg bw/day in parental females) based on the decreased pup viability which was likely to be subsequent to insufficient maternal care. 

In a prenatal developmental toxicity study (OECD Guideline 414), the test substance 2 -imidazolidone was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity (BASF SE, 2018).

Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle.

Regarding clinical examinations, signs of maternal toxicity were observed at the highest test group (1000 mg/kg bw/d). The mean food consumption of the test group 3 dams (1000 mg/kg bw/d) was statistically significantly reduced at the end of gestation (GD 19-20; -15% in comparison to the concurrent control). If calculated for the entire treatment (GD 6-19), the high- dose dams consumed about 4% less food than the controls. The body weight change of the high-dose dams was decreased compared to control (around -10%, without statistical significance) over the treatment period (GD 6-19). The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of test group 3 (1000 mg/kg bw/d) was statistically significantly reduced (-13% compared to control). Insummary, together with the reduction in food consumption,the decrease in(corrected) body weight (change/gain) in the high-dose dams was assessed as treatment-related and adverse. The low- and mid-dose dams (40 and 200 mg/kg bw/d) showed no treatment-related, adverse findings.

Regarding pathology, no treatment-related findings were noted. The single macroscopic find- ing occurred individually. It was considered to be incidental or spontaneous in origin and without any relation to treatment.

No differences of toxicological relevance between the control and the treated groups (40, 200 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose.

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on toxicity to reproduction, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).