Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): EXP 3982 (N-2-hydroxyethylurea)
- Physical state: not reported
- Analytical purity: aqueous solution containing 57.58% hydroxyethyl urea
- Impurities (identity and concentrations): not reported
- Composition: aequeous solution containing 57.58 % hydroxyethyl urea
- Purity test date: not reported
- Lot/batch No.: not reported
- Expiration date of the lot/batch: not reported
- Storage condition of test material: at room temperature in the dark

Test animals

Species:
mouse
Strain:
other: Crl:CD-1 (lCR) BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina (dose rangefinding study) or Kingston, New York (micronucleus assay)
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 30.5-34.5 g (males, dose range finding), 21.8 - 26.7 g (females, dose range finding), 29.4 - 35.3 g (micronucleus assay)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in groups of up to 5 animals in sanitary polycarbonate cages containing bedding
- Diet (e.g. ad libitum): PMI Feeds, Inc., Certified Rodent Diet # 5002 ad libitum
- Water (e.g. ad libitum): tap water ad liblitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 - 26.1
- Humidity (%): 30-70
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2001-07-06 to 2001-08-29

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: not reported
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prior to dosing, the top stock of the test article, EXP 3982 (N-2-Hydroxyethylurea), was prepared by adding the appropriate volume of the vehicle, cell culture grade (deionised) water, to a pre-weighed quantity of the test article, forming transparent, colourless solution. All stocks were prepared by adjusting for a correction factor of 1.74 to account for the content of hydroxyethyl urea of 57.58% in the test substance.
Duration of treatment / exposure:
single oral dose
Frequency of treatment:
single oral dose
Post exposure period:
500 mg/kg: 24 hour treatment
1000 mg/kg: 24 hour treatment
2000 mg/kg: 24 and 48 hour treatment
Vehicle control: 24 and 48 hour treatment
Positive control: 24 hour treatment
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
dose range finding: 500, 1000, 2000 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
micronucleus assay, 24 h preparation interval: 500, 1000, 2000 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
micronucleus assay, 48 h preparation interval: 2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Dose range finding assay: 3 males and 3 females per dose
Micronucleus assay: 6 males per dose and harvest timepoint
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): not reported, but substance proposed in OECD TG 474
- Route of administration: oral gavage
- Doses / concentrations: 80 mg/kg

Examinations

Tissues and cell types examined:
- clinical signs: examination for toxic signs and mortalities about 1 hour after dosing, and at least daily for the duration of this assay
- bone marrow cells: sampling at 24 or 48 hours after treatment
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a dose range finding study, all animals were examined immediately after dosing, about 1 hour after dosing, and at least daily for the duration of this assay for toxic signs and/or mortality. All animals appeared normal immediately after dosing and remained healthy until the end of the observation period. Based on these results, dose levels on the basis of the active ingredient of the test article of 500, 1000, and 2000 mg/kg were selected for for the micronucleus assay. Only males were used in the micronucleus assay because there were no substantial differences in clinical observations between the sexes in the dose rangefinding assay.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest timepoints, the animals were euthanised by CO2 inhalation followed by incision of the diaphragm. The hind limb bones were removed for marrow extraction from five surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal). Animals not needed for bone marrow collection were euthanised at the completion of the assay. Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, stained in May-Grunwald solution followed by Giemsa, and protected by permanently mounted coverslips. For control of bias, all slides were coded prior to analysis.

METHOD OF ANALYSIS:
Slides prepared from the bone marrow collected from five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed scoring at
least the first 500 erythrocytes per animal. Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1120 and 115 the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than 1 micronucleus was counted as 1 micronucleated PCE, not 2 (or more) micronuclei .
Evaluation criteria:
The criteria for a positive response is the detection of a statistically significant positive response for at least one dose level and a statistically significant dose-related response. A test article that does not induce both of these responses will be considered negative. Equivocal results may require further investigation. Statistical significance will not be the only determinant of a positive response, and the study director will consider the biological relevance of the results in the final evaluation.
Statistics:
Assay data analysis will be performed using an analysis of variance (Winer, 1971) on untransfomed proponions of cells with micronuclei per animal when the variances are homogeneous. Ranked proportions will be used for heterogeneous variances. If the analysis of variance is significant (p <= 0.05), a Dunnett's t-test will be used to determine which dose groups, if any, are significantly different from the vehicle control. Analyses will be performed separately for each sampling time. Additionally, parametric or nonparametric tests for trend may be employed to identify any dose-related response.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
, but tested up to limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 1000 mg/kg
- Solubility: Soluble at all doses
- Clinical signs of toxicity in test animals: no
- Evidence of cytotoxicity in tissue analysed: no tissue analysed
- Rationale for exposure: It is recommended in the guideline OECD 474 to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. Two dose levels with an approximate spacing factor of 2 were chosen below this limit dose.
- Harvest times: no harvest in dose range finder
- Gender specific differences in toxicity: no

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): A statistically significant increase in micronucleated PCEs was not observed at any dose level or harvest timepoint.
- Ratio of PCE/NCE (for Micronucleus assay): no statistically significant decrease in the PCE:NCE ratio
- Appropriateness of dose levels and route: tested up to the highest recommended concentration

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test article, EXP 3982 (N-2-Hydroxyethylurea), was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.
Executive summary:

The objective of this study was to evaluate the test article, EXP 3982 (N-2-Hydroxyethylurea), for in vivo clastogenic activity and/or disruption of the mitotic apparatus by quantifying micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1 (ICR) BR mouse bone marrow. The study was conducted under GLP according to the method outlined in OECD TG 474.

In the dose rangefinding assay, the test article was dissolved in cell culture grade (deionised) water and dosed by oral gavage to three males and three females per dose level. The concentration of hydroxyethyl urea in the test material was 57.58%. All stocks were prepared by adjusting for a correction factor of 1.74. The animals were dosed on the basis of the active ingredient of the test article at 500, 1000, and 2000 mg/kg and observed for up to 2 days after dosing for toxic signs and/or mortality.

Based on the results of the dose rangefinding assay, the maximum tolerated dose was estimated to be 2000 mg/kg. In the micronucleus assay, the test article was dissolved in cell culture grade (deionised) water and dosed by oral gavage to six males per dose level at each harvest timepoint. The animals were dosed at 500, 1000, and 2000 mg/kg based on hydroxyethyl urea. Five animals dosed with the test article at 500 and 1000 mg/kg dose levels and five animals dosed with the positive control article were euthanised approximately 24 hours after dosing for extraction of the bone marrow. Five animals dosed with the test article at the 2000 mg/kg dose level and five animals dosed with the vehicle control article were euthanised approximately 24 and 48 hours after dosing for extraction of the bone marrow. At least 2000 PCEs per animal were analysed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal.

The test article, EXP 3982 (N-2-Hydroxyethylurea), induced no signs of clinical toxicity in any of the treated animals and was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio). A statistically significant increase in micronucleated PCEs was not observed at any dose level or harvest timepoint. The test article, EXP 3982 (N-2-Hydroxyethylurea), was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.