Registration Dossier

Administrative data

Description of key information

In a well-conducted subacute oral toxicity study, no NOAEL could be derived. The LOAEL was 1000 ppm (equal to 76 mg/kg bw/day in males and 92 mg/kg bw/day in females) based on hypertrophy of the follicular epithelium of the thyroid glands in two males and one female and thickening of the mucosa of the duodenum in all males and one female.

In a well-conducted combined repeated dose toxicity study with the reproduction/toxicity screening test, the NOAEL was 500 ppm based on decreased body weight/body weight gain, decreased food consumption, and the occurrence of hypertrophy/ hyperplasia in the thyroid glands in males and females of test group 3 (2000 ppm).

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 thyroid gland findings and impaired body weight development into account. In this study, the NOAEL was 100 ppm in male (8.3 mg/kg bw/d) and in female (10.5 mg/kg bw/d) Wistar rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
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
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
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.
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
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
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%).
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.
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
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
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
8.3 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
endocrine system
Organ:
thyroid gland

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an OECD Guideline 408 study, Ethylene Urea was administered via drinking water to groups of 10 male and 10 female Wistar rats at concentrations of 0 ppm (test group 0), 100 ppm (test group 1), 500 ppm (test group 2) and 2000 ppm (test group 3) over a period of 3 months. Drinking water served as vehicle.

In addition to the required examinations, special attention was given to the reproductive organs of male and female animals.

 Food consumption, drinking water consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and towards the end of the administration period. For at least 3 weeks an estrous cycle determination was performed. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out towards the end of the administration period.

Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations. Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals for spermexaminations.

The following test substance-related, relevant findings were noted:

Test group 3: 2000 ppm (males: 148 mg/kg bw/d, females: 186 mg/kg bw/d)

Clinical Examinations

- Mean body weights were lower in male and female animals during the entire administration period, with a maximum of -6.8% on study day 63 in males (significant) and -5.3% on study day 70 in females (notsignificant).

- The mean body weight change values were lower in male and female animals during the entire administration period, with a maximum of -12.7% between study days 0 and 28 in males (significant) and -12.5% between study days 0 and 70 in females (not significant).

Clinical Pathology

- No treatment-related, adverse effects were observed.

Pathology

- Significant increase of thyroid glands, i.e. +43% (absolute) and +51% (relative) in males and +35% (absolute) and +41% (relative) in females in combination with minimal to moderate hypertrophy/hyperplasia in all males and nine females.

Test group 2: 500 ppm (males: 39 mg/kg bw/d, females: 48 mg/kg bw/d)

Clinical Examinations and Clinical Pathology

- No treatment-related, adverse effects were observed.

Pathology

- Minimal to slight hypertrophy/hyperplasia in thyroid glands of five males and six females

Test group 1: 100 ppm (males: 8.3 mg/kg bw/d, females: 10.5 mg/kg bw/d)

Clinical Examinations, Clinical Pathology and Pathology

- No treatment-related, adverse effects wereobserved.

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 rat.

2 -week mechanistical study

Ethylene Urea has shown a potential to affect rat thyroid morphology. Possibly, an indirect mechanism may be related in an increased clearance of thyroid hormones by liver enzymes. In order toelucidate the mechanism by which the test substance causes thyroid toxicity in rats, liver enzyme induction and activity were examined after treatment with 2-Imidazolidone.

Ethylene Urea was administered via the drinking water to groups of 10 male and 10 female Wistar rats at concentrations of 0 (test group 0), 500 (test group 1) and 2000 ppm (test group 2) over a period of 2 weeks. 

Food consumption, water consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.

Hormones level (T3, T4, TSH) analyzes were performed on study day -3, 3, 7 and 14 from all animals. After the administration period, all rats were sacrificed and assessed by gross pathology. Liver and thyroid gland weights were determined. Parts of the liver tissue were fixed, others were deep frozen in liquid nitrogen for bioanalytical examinations.

For bioanalytical examinations, liver microsomes were prepared and characterized for their total cytochrome P450-content as well as for the activities of Ethoxyresorufin-O-deethylase (EROD), Pentoxyresorufin-O-depentylase (PROD), Benzyloxyresorufin-O-debenzylase (BROD), 4-Methylumbeliferone-glucuronyltransferase (MUF-GT), 4-Hydroxybiphenyl-glucuronyltransferase (HOBI-GT) and T4-specific UDP-glucuronosyltransferase.

Test group 2: 2000 ppm (124 mg/kg bw/d in males and 195 mg/kg bw/d in females)

Clinical Examinations

- No treatment-related effects were observed. Clinical Pathology

Clinical Pathology

- Decreased T4 levels in both sexes on study days 3, 7 and 14

- Increased TSH levels in both sexes on study days 3, 7 and 14

- Decreased T3 levels in males on study days 3, 7 and 14

- Decreased T3 levels in females on study day 3, only

Pathology

- Significantly increased absolute (+42%) and relative (+41%) thyroid gland weights in male animals

Bioanalytical Examinations

- Increased totalcytochrome P450-contentin females

- Increased PROD-activity in males

- Increased MUF-GT-activity in males and females

- Increased HOBI-GT-activity in males

  

Test group 1: 500 ppm (34 mg/kg bw/d in males and 44 mg/kg bw/d in females)

Clinical Examinations

- No treatment-related effects were observed.

Clinical Pathology

- Decreased T4 levels in both sexes on study days 3, 7 and 14

- Increased TSH levels in both sexes on study days 3, 7 and 14

- Decreased T3 levels in males on study day 14

Pathology

- Significantly increased absolute (+34%) and relative (+34%) thyroid gland weights in male animals

Bioanalytical Examinations

- Increased EROD-activity in females

- Increased HOBI-GT-activity in males and females

- Increased T4-GT-activity in females

 

In conclusion, the treatment of male and female Wistar rats with Ethylene Urea via drinking water for 2 weeks resulted in reducedthyroid hormone levels in both sexes, increased thyroid weights in male animals and the induction of liver enzymes in both sexes.

Decreased thyroid hormone levels were found in both test groups, i.e. at 500 and 2000 ppm, in combination to increased TSH levels. Thyroid gland weights were increased also in both test groups but only in male animals.

The total cytochrome P450 content in liver was increased in female animals of the high-dose group (2000 ppm). PROD activity was increased in male animals of the high-dose group and EROD activity was increased in female animals of the low-dose group (500 ppm).

Glucuronosyltransferases were induced by the treatment as demonstrated by an increase of MUF-GT in male and female animals of the high-dose group and HOBI-GT in male and female animals of the low-dose group. HOBI-GT was also increased in male animals of the high-dose group. T4-GT was increased in female animals of the low-dose group.

In a GLP compliant OECD guideline 422 study, Ethylene Urea 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 this 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.

The NOAEL (no observed adverse effect level) for general, systemic toxicity was 500 ppm (37 mg/kg bw/day in parental males and 57 mg/kg bw/day in parental females), based on the decreased body weight/body weight gain and decreased food consumption.

 

In an oral subacute toxicity study performed under GLP and according to OECD guideline 407, Wistar rats (5/sex/dose) were exposed daily for four weeks to the substance in drinking water at concentrations of 0 ppm (vehicle control), 1000 ppm, 4000 ppm and 12000 ppm (equal to 76, 288 and 724 mg/kg bw/day in males and 92, 362 and 1002 mg/kg bw/day in females) (BASF AG, 2002).

Food consumption, water consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. A functional observational battery and measurement of motor activity was carried out after 4 weeks of treatment. Clinicochemical, hematological examinations and urinalyses were carried out towards the end of the administration period. The animals were assessed by gross pathology, followed by histopathological examinations.

The following substance related findings were obtained:

1000 ppm:

- hypertrophy of the follicular epithelium of the thyroid gland (2 males, 1 female)

- thickening of the mucosa of the duodenum in all animals

4000 ppm:

both sexes:

- decreased food and water consumption and impairment of body weight

- increases in urinary ketones

- hypertrophy of the follicular epithelium in the thyroid glands

- hyperplasia of the follicular epithelium of the thyroid glands (2 males, 4 females)

- thickening of the mucosa of the duodenum (5 males, 2 females)

in males only:

- impaired food efficiency

- decreases in inorganic phosphate and urinary volume

- increase in urinary specific gravity

- testicular tubular giant cells and tubular hypoplasia (1 male)

- cellular debris in the epididymides (1 male)

12000 ppm:

both sexes:

- anogenital regions smeared with urine, piloerection, and/or reduced general state

- decreased food and water consumption, impairment of body weight and food efficiency

- decreases in red blood cells, hemoglobin, hematocrit, alkaline phosphatase and triglycerides

- increase in microcytosis, anisochromia, chloride and urinary ketones

- hypertrophy and hyperplasia of the follicular epithelium of the thyroid glands

- focal liver cell necrosis (3 males, 2 females)

- bile duct proliferation (3 males, 3 females)

- thickening of the mucosa of the duodenum (all males, 4 females)

- development of germinal centers in the spleen (5 males, 4 females)

- development of germinal centers in the mandibular lymph nodes (4 males, 4 females)

- development of germinal centers in the mesenteric lymph nodes (5 males, 3 females)

in males only:

- decreases in inorganic phosphate, calcium and urinary volume

- increase in urinary specific gravity

- dark yellow discoloration and cloudy appearance of the urine specimens

- testicular tubular giant cells and tubular hypoplasia (2 males)

- cellular debris in the epididymides (all males)

- hyperplasia of the urothelium in the urinary bladder (3 males), associated with focal metaplasia to squamous epithelium (1 male)

in females only:

- lymphocyte depletion in the thymus and hypocellularity of the bone marrow (1 female)

- many clear cell foci in the liver (1 female)

- increased severity of the hemosiderin deposition in the spleen

Thus, toxic effects were seen at all dose levels. Target organs were thyroid glands, liver, testes, epididymides, duodenum, urinary bladder, spleen and lymph nodes. Based on this study no NOAEL can be derived. The LOAEL is 1000 ppm (equal to 76 mg/kg bw/day in males and 92 mg/kg bw/day in females).

 

In an old study (non-GLP and not according to current guidelines), four rabbits were exposed up to 6 doses of 2.5 g/kg bw (by gavage) of the substance in 25% aqueous solution (BASF AG, 1959).Two of four animals died after 4 and 5 applications, respectively. The other 2 animals survived 6 treatments. Symptoms included cachexia, diarrhea and impairment of balance. Regarding clinical signs and pathology, no adverse effects were observed. Few erythrocytes in urine and swelling of the kidneys in one of the surviving rabbits and a high quantity of red blood cells in urine in one of the animals which died were observed.

In another old study (non-GLP and not according to current guidelines), seven rabbits were exposed up to 16 doses of 0.9 g/kg bw (by gavage) of the substance in 9% aqueous solution (BASF AG, 1959).Symptoms included apathy, diarrhea, impairment of balance and refusal of food intake. Four animals died after 4 applications, 2 animals after 5 - 8 applications and 1 animal after 16 applications. Regarding clinical signs and pathology, no adverse effects were observed. High levels of urea found in blood are to be interpreted in terms of metabolic nitrogen from the test substance (according to the authors of the study report).

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.

There is evidence from repeated-dose studies that the registered substance causes thyroid enlargement (hypertrophy/hyperplasia) when administered to rats in the drinking water. To further investigate the mode of action of thyroid effects, a 14-days in vivo mechanistic study was performed in rats with additional thyroid investigations (determination of thyroid hormones (total triiodothyronine (T3) and thyroxine (T4)) and thyroid stimulating hormone (TSH)) and assessment of liver enzyme amounts/activities.

The results of this mechanistic study support the hypothesis that thyroid enlargement is probably related to increased TSH drive in response to decreases in circulating plasma levels of T3 and T4. Furthermore, an induction of liver enzymes in both sexes was observed. However, induction effects, especially for T4-GT, were assessed to be weak when compared to effects of well-studied enzyme inducers leading to increased clearance of T4 (Liu et al., 1995). Therefore, the results of the mechanistic investigations do not clearly prove that induction of specific Phase II enzymes responsible for hepatic clearance of T3 and T4 is the exclusively operating Mode of Action (MoA) of thyroid toxicity for the registered substance.

 

In conclusion, based on available data on repeated dose toxicity and mechanistic investigations to further investigate the MoA of the observed thyroid toxicity, the test item is classified STOT RE cat. 2 (thyroid gland) according to Regulation (EC) No 1272/2008 (CLP).

 

References:

Liu J, Liu Y, Barter RA, Klaassen CD (1995) Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study. J Pharmacol Exp Ther 273: 977-985.