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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-28-2016 - 11-15-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
The analyses of the test item (= test substance) was carried out at Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany (study code 16L00102).

Name of test substance: 2-Imidazolidone
CAS No. 120-93-4
Test substance No: 08/0054-5
Batch identification: 16960025
Identity: Confirmed
Purity: 89.2 corr.area-% (GC) (study code:16L00102)
Homogeneity: Given
Stability: Stable until 28 Feb 2018
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
The test facility is organizationally independent from the BASF SE sponsor division.

ADDITIONAL TEST-SUBSTANCE INFORMATION
Synonym: Ethylene Urea
Physical state/appearance: Solid/white
Storage conditions: Ambient

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA.
Sex:
male/female
Details on test animals and environmental conditions:
The animals were housed together (5 animals per cage) in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Typ NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, and large play tunnels (Art. 14153) supplied by PLEXX B.V., Elst, The Netherlands, were added for environmental enrichment. The animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24°C, a range of relative humidity of 30-70% and 15 air changes per hour. The day/night cycle was 12 hours (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h). There were no or only minimal deviations from these limits. The animal room was completely disinfected prior to the study using a disinfector ("AUTEX", fully automatic, formalin-ammonia-based terminal disinfector). The floor and the walls 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 (from water bottles) were available ad libitum.

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as an aqueous 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 volume and subsequently homogenized with a magnetic stirrer. The test-substance preparations were produced twice a week and stored at room temperature.

VEHICLE: drinking water
- Concentration in vehicle (drinking water):
100, 500 and 1000 ppm
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, 67056 Ludwigshafen, Germany.
The stability of the test substance in drinking water at room temperature for a period of 4 days was proven in a comparable batch before the start of the study (study code 01Y0054/08Y010).

The test substance is completely miscible with drinking water and thus an aqueous solution. Therefore, the test substance preparation was considered to be homogenous. Consequently, homogeneity analysis was not carried out.
Moreover, concentration control analyses were performed in all concentrations at the beginning as well as towards the end of the study.

The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 94- 100% of the nominal concentrations. Taken together, the results demonstrate the correctness of the concentrations of 2-Imidazolidone.
Duration of treatment / exposure:
90 days
Frequency of treatment:
ad libitum
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm
Remarks:
8.3 and 10.5 mg/kg bw/d in males/females
Dose / conc.:
500 ppm
Remarks:
39 and 48 mg/kg bw/d in males/females
Dose / conc.:
2 000 ppm
Remarks:
148 and 186 mg/kg bw/d in males/females
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard. Doses were selected to acertain comparability to the previously conducted OECD 422 guideline repeated dose study (88R0054/08C017) and the contemporaneous conducted 2 weeks mechanistic study (99C0054/08S041) on the same test compound.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
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.

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were checked daily for any abnormal clinical signs. Abnormalities and changes will be documented for each animal.
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:

1.Abnormal behavior when handled
2.Fur
3.Skin
4.Posture
5.Salivation
6.Respiration
7.Activity/arousal level
8.Tremors
9.Convulsions
10.Abnormal movements
11.Impairment of gait
12.Lacrimation
13.Palpebral closure
14.Exophthalmus
15.Feces (appearance/ consistency)
16.Urine
17.Pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION: Yes
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Water consumption was determined weekly (as representative value over 4 days; Thursday- Monday) and calculated as mean water consumption in grams per animal and day.
The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and mean water consumption per cage.
(WCx * C) / BWx = test substance intake for study day x; BWx = body weight on study day x [g], WCx = mean daily water consumption on study day x [g], C = concentration in drinking water on study day x [mg/kg]

OPHTHALMOSCOPIC EXAMINATION: Yes
Prior to the start of the administration period on day -3 the eyes of all animals and on study day 84 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

HAEMATOLOGY: Yes
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out 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 following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
Parameters and methods:
Parameter Unit Method
Leukocyte count (WBC) giga/L cytochemistry coupled with flow cytometry
Erythrocyte count (RBC) tera/L flow cytometric laserlight scattering
Hemoglobin (HGB) mmol/L cyanmethemoglobin method; according to ICSH
Hematocrit (HCT) L/L calculation:MCV x erythrocytes
Mean corpuscular volume
(MCV) fL RBC/PLT method; mean of RBC volume distribution curve (histogram)
Mean corpuscular
hemoglobin (MCH) fmol calculation:hemoglobin/erythrocytes
Mean corpuscular hemoglobin
concentration (MCHC) mmol/L calculation:hemoglobin/hematocrit
Platelet count
(PLT) giga/L flow cytometric laserlight scattering
Differential blood count % and giga/L cytochemistry coupled with flow cytometry
Reticulocytes
(RETA) giga/L cytochemistry coupled with flow cytometry

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). Only evaluated blood smears were archived.

Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).

CLINICAL CHEMISTRY: Yes
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out 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.
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters

Parameters and methods:

Enzyme (systematic name and system number) Unit Method, wave- length and measuring
temperature (Detection limit)
Alanine aminotransferase (ALT)
(L-alanine: 2-oxoglutarate aminotransferase;
EC 2.6.1.2.) μkat/L kinetic UV test, 340 nm; 37°C, (0.08 μkat/L)
Aspartate aminotransferase (AST)
(L-aspartate: 2-oxoglutarate aminotransferase;
EC 2.6.1.1.) μkat/L kinetic UV test, 340 nm; 37°C, (0.08 μkat/L)
Alkaline phosphatase (ALP)
(orthophosphoric acid monoester phosphohydrolase;
EC 3.1.3.1.) μkat/L kinetic color test, 415 nm, 37°C, (0.084 μkat/L)
gamma-Glutamyltransferase (GGT)
(gamma-glutamyl) peptide: aminoacid-gamma- glutamyl-transferase;
EC 2.3.2.2.) nkat/L kinetic color test, 415 nm, 37°C, (25 nkat/L)
Sodium (NA) mmol/L
Potassium (K) mmol/L ion selective electrodes (ISE), (Na: 80, K: 1.5, Cl: 60 nmol/L)
Chloride (CL) mmol/L
Inorganic phosphate (INP) mmol/L molybdate reaction (0.1 mmol/L)
Calcium (CA) mmol/L mmol/L o-cresolphthalein complex without deproteinization (0.2 mmol/L)
Urea (UREA) mmol/L mmol/L enzymatic determination with the urease/ glutamate dehydro- genase method (0.5 mmol/L)
Creatinine (CREA) μmol/L enzymatic determination with the creatininase/ creatinase /sarcosinoxidase method (5 μmol/L)
Glucose (GLUC) mmol/L hexokinase/glucose-6-phosphate dehydrogenase method (0.11 mmol/L)
Total bilirubin (TBIL) μmol/L DPD method (0.56 μmol/L)
Total protein (TPROT) g/L biuret method (2 g/L)
Albumin (ALB) g/L bromocresol green method (3.2 g/L)
Globulins (GLOB) g/L difference between total protein and albumin
Triglycerides (TRIG) mmol/L enzymatic color test with lipase esterase/ glycerokinase/ glycerol- 3-phosphate oxidase/4-amino- phenazone (0.1 mmol/L)
Cholesterol (CHOL) mmol/L enzymatic determination with cholesterol esterase/ cholesterol oxidase/4-amino-phenazone (CHOD-PAP method) (0.1 mmol/L)

URINALYSIS: Yes
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 dry chemical reactions on test strips (Combur-Test 10 M; Sysmex, Norderstedt, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Sysmex, Norderstedt, Germany).

Parameters and methods
Parameter Method
pH methyl red and bromothymol blue
Protein (PRO) tetrabromophenol-phthaleinethylester (TBPE)
Glucose (GLU) GOD-POD reaction
Ketones (KET) sodium nitroprusside
Urobilinogen (UBG) p-methoxyaniline-diazonium-salt
Bilirubin (BIL) 2,5-dichloroaniline diazonium salt
Blood 2,5-dimethylhexane-2,5-dihydroperoxide, tetramethylbenzidine
Specific gravity
(SP.GR.) [g/L] refractometer
Sediment microscopy
Color, turbidity (COL, TURB) by visual evaluation
Volume (VOL) [mL] graduated tubes

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random. A detailed description of the methods, the ranking and documentation system can be found in PART III (Supplement).

Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the rats. Attention was paid to:
1.Posture
2.Tremors
3.Convulsions
4.Abnormal movements
5.Gait abnormalities
6.Other findings


Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:
1.Behavior on removal from the cage
2.Fur
3.Skin
4.Salivation
5.Nasal discharge
6.Lacrimation
7.Eyes/ pupil size
8.Posture
9.Palpebral closure
10.Respiration
11.Tremors
12.Convulsions
13.Abnormal movements/ stereotypes
14.Gait abnormalities
15.Activity/ arousal level
16.Feces excreted within 2 minutes (appearance/ consistency)
17.Urine excreted within 2 minutes (amount/ color)
18.Rearing within 2 minutes
19.Other findings


Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:
1.Reaction to an object being moved towards the face (approach response)
2.Touch sensitivity (touch response)
3.Vision (visual placing response)
4.Pupillary reflex
5.Pinna reflex
6.Audition (auditory startle response)
7.Coordination of movements (righting response)
8.Behavior during handling
9.Vocalization
10.Pain perception (tail pinch)
11.Grip strength of forelimbs
12.Grip strength of hindlimbs
13.Landing foot-splay test
14.Other findings


Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the rats during these measurements and the measurement room was darkened after the transfer of the last rat. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.


IMMUNOLOGY: No

OTHER:
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.

Sperm parameters
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male on schedule. Sperm motility examinations were carried out in a randomized sequence.
Sperm morphology and sperm head count (testis and cauda epididymis) were determined in the control and highest test group, only.

Parameters and methods:
Parameter Unit Method
Sperm motility % microscopic evaluation
Sperm morphology % vital staining with eosin; microscopic evaluation
Sperm head count (cauda epididymis) Mio/g cauda epididymis microscopic evaluation with MAKLER chamber after homogenization
Sperm head count (testis) Mio/g testis microscopic evaluation with MAKLER chamber after homogenization

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy:
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights:
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals
2.Adrenal glands
3.Brain
4.Cauda epididymis
5.Epididymides
6.Heart
7.Kidneys
8.Liver
9.Ovaries
10.Pituitary gland
11.Prostate
12.Seminal vesicles with coagulating glands
13.Spleen
14.Testes
15.Thymus
16.Thyroid glands
17.Uterus with cervix

Organ/tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1.All gross lesions
2.Adrenal glands
3.Aorta
4.Bone marrow (femur)
5.Brain
6.Cecum
7.Cervix
8.Coagulating glands
9.Colon
10.Duodenum
11.Epididymis, left (modified Davidson’s solution)
12.Esophagus
13.Extraorbital lacrimal glands
14.Eyes with optic nerve (modified Davidson’s solution)
15.Femur with knee joint
16.Harderian glands
17.Heart
18.Ileum
19.Jejunum (with Peyer’s patches)
20.Kidneys
21.Larynx
22.Liver
23.Lungs
24.Lymph nodes (mesenteric and axillary lymph nodes)
25.Mammary gland (male and female)
26.Nose (nasal cavity)
27.Ovaries
28.Oviducts
29.Pancreas
30.Parathyroid glands
31.Pharynx
32.Pituitary gland
33.Prostate
34.Rectum
35.Salivary glands (mandibular and sublingual glands)
36.Sciatic nerve
37.Seminal vesicles
38.Skeletal muscle
39.Skin
40.Spinal cord (cervical, thoracic and lumbar cord)
41.Spleen
42.Sternum with marrow
43.Stomach (forestomach and glandular stomach)
44.Testis, left (modified Davidson’s solution)
45.Thymus
46.Thyroid glands
47.Trachea
48.Urinary bladder
49.Uterus
50.Vagina

The right testis and epididymis were used for sperm parameters


HISTOPATHOLOGY: Yes
The histotechnical processing (trimming, paraplast embedding, cutting, hematoxylin and eosin (H&E) staining) of all organs of all animals (according to the study plan, the amendments to the study plan and the BASF raw data) were accomplished by the test site Propath UK Limited, Willow Court, Netherwood Road, Hereford, HR2 6JU, UK, under the responsibility of the Principal investigator (PI) Mrs. Nicola Lewis. Raw data of the study phase, as well as remaining wet tissue, paraplast blocks and HE-stained slides were sent to the test facility for archiving for at least the period of time specified in the GLP principles. Histopathological evaluation of the HE-stained slides was performed by the study pathologist of BASF SE.

Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:

Organs Test group
0 1 2 3
1. All gross lesions A2 A2 A2 A2
2. Adrenal glands A1 A1
3. Aorta A1 A1
4. Bone marrow (femur) A1 A1
5. Brain A1 A1
6. Cecum A1 A1
7. Cervix A1 A1
8. Coagulating glands A1 A1
9. Colon A1 A1
10. Duodenum A1 A1
11. Epididymis, left A1 A1
12. Esophagus A1 A1
13. Eyes with optic nerve A1 A1
14. Female mammary gland A1 A1
15. Heart A1 A1
16. Ileum A1 A1
17. Jejunum A1 A1
18. Kidneys A1 A1
19. Liver A1 A1
20. Lung A1 A1
21. Lymph nodes
(mesenteric and axillary lymph nodes) A1 A1
22. Ovaries A1 A1
23. Pancreas A1 A1
24. Parathyroid glands A1 A1
25. Peyer’s patches A1 A1
26. Pituitary gland A1 A1
27. Prostate A1 A1
28. Rectum A1 A1
29. Salivary glands
(mandibular and sublingual glands) A1 A1
30. Sciatic nerve A1 A1
31. Seminal vesicles A1 A1
32. Skin A1 A1
33. Spinal cord
(cervical, thoracic and lumbar cord) A1 A1
34. Spleen A1 A1
35. Stomach
(forestomach and glandular stomach) A1 A1
36. Testis, left A1 A1
37. Thymus A1 A1
38. Thyroid glands A1 A1 A1 A1
39. Trachea A1 A1
40. Urinary bladder A1 A1
41. Uterus A1 A1
42. Vagina A1 A1

A = Hematoxylin and Eosin (H&E) stain 1 = all animals/test group
2 = all animals affected/test group

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).

A correlation between gross lesions and histopathological findings was attempted.
Statistics:
Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
Rearing, grip strength forelimbs, grip strength hindlimbs, foot- splay test, motor activity, estrous cycle: 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.
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians.
Urinalysis parameters (apart from pH, urine volume and specific gravity, color and turbidity): Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. In case of exactly the same numbers of the dose group and the control, no statistical test is performed.
Urine pH, volume and specific gravity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians.
Urine color and turbidity: not evaluated statistically.
Sperm analysis parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians; If only control and one dose group are measured, WILCOXON-test (one-sided) without adjustment were used.For the percentage of abnormal sperms (ABNORMAL6_C) values < 6% were set to 6% (cut off 6%).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related impairment of body weight parameters were observed in male and female animals in test group 3 (2000 ppm).

Mean body weight of male animals in test group 3 (2000 ppm) was lower during the entire administration period, showing a maximum and significant deviation to the control of -6.8% on study day 63. Body weight of female animals in test group 3 (2000 ppm) was lower (although not significantly) during the entire administration period, showing a maximum deviation to the control of -5.3% on study day 70.
No treatment-related changes in mean body weights were observed for male and female animals of test groups 1 and 2 (100 and 500 ppm).

Mean body weight change values were significantly decreased in male animals of test group 3 (2000 ppm) during the entire administration period except study day 91, showing a maximum deviation to the control of -12.7% between study days 0 and 28. In female animals of test group 3 (2000 ppm) body weight change values were also lower during the entire administration period. Statistical significance was only given between study days 0 and 77 (-11.7%), maximum deviation to the control was achieved by -12.5% between study days 0 and 70.
No treatment-related changes in mean body weight change values were observed for male and female animals of test groups 1 and 2 (100 and 500 ppm).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to food consumption were observed.
Although food consumption values of male and female animals in test group 3 (2000 ppm) were lower compared to the control values on several days of the administration period, they were all within the range typical for rats of this strain and age.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to water consumption were observed.
Although water consumption values of male and female animals in test group 3 (2000 ppm) were lower compared to the control values on several days of the administration period, they were all within the range typical for rats of this strain and age.

Intake of test substance:
The mean daily test substance intake in mg/kg body weight/day (mg/kg bw/d) over the entire study period was calculated and is shown in the following table:

Test group Concentration in drinking water (ppm) Mean daily test-substance intake (mg/kg/d)
Males Females Males Females
1 100 100 6.9 8.5
2 500 500 34.4 48.1
3 2000 2000 138.0 152.7
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were observed.
All apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
After the administration period in males of test group 3 (2000 ppm) platelet counts were significantly higher compared to controls, but the mean was within the historical control range (males: platelets 643-836 Giga/L). In females of test group 1 (100 ppm) red blood cell (RBC) counts were significantly lower compared to controls, but the change was not dose- dependent. Therefore, both alterations were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
After the administration period in males of test group 3 (2000 ppm) chloride levels were significantly higher compared to control, but the mean was within the historical control range (males: chloride 98.8-105.9 mmol/L). In males of test group 2 (500 ppm) alanine aminotransferase (ALT) activities were significantly decreased, but the change was not dose-dependent. Therefore, both mentioned alterations were regarded as incidental and not treatment-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
After the administration period in the urine of rats of both sexes of test group 3 (2000 ppm) the incidence of ketone bodies was significantly increased. Because no indication of a dysregulation of the energy metabolism was present in these individuals, this finding was most probably due to an interference with excreted compound or some of its metabolites by the kidneys. Therefore, this alteration was regarded as treatment-related, but not adverse.
In males of test groups 1, 2 and 3 (100, 500 and 2000 ppm) urine volume was significantly lower compared to controls. However, without any other finding in the renal system this effect reflected the concentration capacity of the kidneys and was regarded as adaptive and non-adverse. In males of test group 1 (100 ppm) higher incidences of triple phosphate crystals were found in the urine sediment and in males of test group 2 (500 ppm) hemoglobin was found in the urine. However, these findings were not dose-dependent and therefore they were regarded as incidental and not treatment-related.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
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 to have been incidental.
The following examinations were performed during FOB and have to be 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:
No test substance-related effects were observed.

Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for male and female animals of test groups 1-3 (100, 500 and 2000 ppm).
At interval Nos. 2 and 3 significantly increased values were measured for female animals of test group 3 (2000 ppm). These deviations were assessed to be spontaneous as the overall activity was not significantly changed.
At interval No 10 a significantly decreased value was measured for female animals of test group 1 (100 ppm). The individual deviation was assessed not to be related to treatment as no dose-response relationship occurred and the overall activity was not significantly changed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute organ weights:
When compared with control group 0 (set to 100%), the following mean absolute weights were significantly changed (statistically significant changes marked with **: p ≤ 0.01):
Male animals Female animals
Test group (ppm) (100) (500) (2000) (100) (500) (2000)
Testes 111%** 106% 103%
Thyroid glands 112% 107% 143%** 102% 104% 135%
All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights (relative to terminal body weight):
When compared with control group 0 (set to 100%), the following mean relative organ weights were significantly changed (statistically significant changes are marked with *: p ≤ 0.05 or **: p ≤ 0.01):
Male animals Female animals
Test group (ppm) (100) (500) (2000) (100) (500) (2000)
Adrenal glands 114% 110% 118%**
Liver 99% 98% 105%*
Testes 110%** 108% 110%*
Thyroid glands 111% 108% 151%** 102% 102% 141%**
All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The absolute and relative weight increases of thyroid glands in males and females of test group 3 (2000 ppm) were regarded to be treatment-related.
The increase of absolute and relative testes weight in test group 1 (100 ppm) was regarded to be incidental due to a missing dose-response relationship and no histopathologic findings in test group 3 (2000 ppm) animals. The increase of relative testes weight in test group 3 (2000 ppm) animals was regarded to be due to the reduced terminal body weight and, therefore, no direct effect on the organ weight.
The increase of relative organ weight of the adrenal glands and the liver of male animals of test group 3 (2000 ppm) were also assumed to have been caused by the reduced terminal body weight in this test group and, therefore, displaying a secondary effect.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the thyroid glands of male and females with incidences and grading according to the table below:

Thyroid glands Male animals Female animals
Test group (ppm) (0) (100) (500) (2000) (0) (100) (500) (2000)
Hypertrophy/
Hyperplasia, diffuse 0 0 5 10 0 0 6 9
Grade 1 2 1 5 2
Grade 2 3 7 1 6
Grade 3 3 1

In the thyroid glands of males and females of test group 2 (500 ppm) and 3 (2000 ppm) a diffuse hypertrophy/hyperplasia was observed. The follicles showed an irregular shape and a much smaller lumen when compared to control animals. This finding was regarded 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
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.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
test mat. (dissolved fraction)
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 ppm
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The administration of 2-Imidazolidone via the drinking water to male and female Wistar rats for 3 months caused test substance-related adverse signs of toxicity at a concentration of 500 ppm and above taking the thyroid gland findings and impaired body weight development into account.
Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 100 ppm in male (8.3 mg/kg bw/d) and in female (10.5 mg/kg bw/d) Wistar rats.
Executive summary:

2-Imidazolidone was administered via drinking water to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 500 (test group 2) and 2000 ppm (test group 3) over a period of 3 months.

With regard to clinical examinations, signs of general systemic toxicity were observed in male and female Wistar rats at a concentration in drinking water of 2000 ppm taking impaired body weight development into account.

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

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a concentration of the compound of 2000 ppm.

Regarding pathology, the thyroid glands were the target organs.

In males and females of test groups 2 (500 ppm) and 3 (2000 ppm) a dose-dependent increase in number of affected animals and severity of follicular hypertrophy and hyperplasia was observed. This was regarded to be caused directly by the test-substance and was therefore assessed as treatment-related and adverse. The increase in thyroid gland weight in test group 3 (2000 ppm) animals was regarded to be caused by the follicular hypertrophy/hyperplasia.

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.