Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 AUG 2010 to 28 SEP 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
During the acclimation phase of the animals used for the pre-experiment the relative humidity was between 45 ¿ 71 % for a maximum of 9 hours.
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
During the acclimation phase of the animals used for the pre-experiment the relative humidity was between 45 ¿ 71 % for a maximum of 9 hours.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
in accordance to German Chemikaliengesetz and Directive 2004/9/EEC
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: OP1
- Expiration date of the lot/batch: October 16, 2014




Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: males mean: 37.6 g (SD +/- 1.8 g)
- Assigned to test groups randomly: yes
- Housing: single in Makrolon cages Type II/III, with wire mesh top
- Diet: pelleted standard diet (Harlan Laboratories B.V., AD Horst, The Netherlands), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-71
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: sterile water
- dose volume: 10 mL/kg bw
- Justification for choice of vehicle: relative non-toxicity.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- test material was dissolved in sterile water on the day of the experiment
- analysis of concentration, homogeneity and stability was not performed

JUSTIFICATION FOR ROUTE OF EXPOSURE:
Oral application is appropriate since Dimethylol dihydroxyethylene urea, a substance with similar structure, is well absorbed from the gastrointestinal tract, through the skin and from the blood circulation, and more than 90% is excreted unchanged in the urine. Thus there is no evidence that the substance, or reactive metabolites, will not reach the target tissue
Duration of treatment / exposure:
0, 500, 1000 and 2000 mg/kg group: 24 hrs
2000 mg/kg group: 48 hrs
Frequency of treatment:
single
Post exposure period:
none
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
7 males per dose and exposure duration
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg
- Exposure duration: 24 hrs

Examinations

Tissues and cell types examined:
bone marrow cells from the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- maximum recommended dose level and two lower dose levels
- since no gender differences occurred in the pre-experiment on toxicity the main study was performed using male animals only

DETAILS OF SLIDE PREPARATION:
- femur was dissected from each animal, aspirated with foetal calf serum and bone marrow smears were prepared following centrifugation and re-suspension. Smears were air-dried and stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
- blind examination with light microscopy (coded slides, NIKON microscopes with 100x oil immersion objectives)
- Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei.
- To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes.
Evaluation criteria:
A test item is classified as mutagenic if:
- it induces either a dose-related increase or
-a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.

A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
nonparametric Mann-Whitney test

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:2000 mg/kg bw, oral
- Clinical signs of toxicity in test animals: neither male nor female animals expressed any toxic reactions


RESULTS OF DEFINITIVE STUDY
GENOTOXICITY
- PCEs with Micronuclei (%):
group mean for control group: 0.114;
- treatment groups did not deviate significantly
- 500 mg/kg bw 24 hrs: 0.107
- 1000 mg/kg bw 24 hrs: 0.071
- 2000 mg/kg bw 24 hrs: 0.121
- 2000 mg/kg bw 48 hrs: 0.093
positive control gave expected result:
- 40 mg/kg bw 24 hrs: 1.829

CYTOTOXICITY
(ratio between PCE and NCE was determined in the same samples and reported as no. of PCEs per 2000 erythrocytes, fesults are presented below)
- control group: 1176
- 500 mg/kg bw 24 hrs: 1113
- 1000 mg/kg bw 24 hrs: 1123
- 2000 mg/kg bw 24 hrs: 1226
- 2000 mg/kg bw 48 hrs: 1172
positive control:
- 40 mg/kg bw 24 hrs: 1122

Any other information on results incl. tables

There was no statistically significant increase in the frequency of micronucleated PCE's and in the PCE/NCE ratio in any dose group when compared to the concurrent vehicle control groups.

The positive control goup showed a marked increase in the incidence of micronucleated PCE.

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-genotoxic under the conditions of this in vivo micronuclei assay.
Executive summary:

Genetic toxicity of the test item has been investigated in an in vivo micronucleus assay in male NMRI mice (7 per dose group). The test item was administered once orally at the maximum recommended dose level of 2000 mg/kg bw, with 1000 and 500 mg/kg bw as the two lower dose levels. Animals were killed 24 or 48 hours later. Evaluation of polychromatic (PCE) or normochromatic (NCE) erythrocytes did not reveal any evidence of an increase in the incidence of micronucleated cells. No significant change in the PCE/NCE ratio was observed after dosing with the test item, and no signs of systemic toxicity were observed in animals dosed with the test item. The positive control material produced a marked increase in the frequency of micronucleated PCE. The test material was considered to be non-genotoxic under the conditions of the test.