1,3-bis(hydroxymethyl)urea

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2012 to 09 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2013

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis(hydroxymethyl)urea

EC Number:

205-444-0

EC Name:

1,3-bis(hydroxymethyl)urea

Cas Number:

140-95-4

Molecular formula:

C3H8N2O3

IUPAC Name:

1,3-bis(hydroxymethyl)urea

Test material form:

solid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: mean value 34.8 g (SD ± 1.7 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: single, Makrolon Type II/III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: ad libitum, pelleted standard diet (Harlan Laboratories B.V.; Postbus 6174; 5960 AD Horst; The Netherlands)
- Water: ad libitum, tap water
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 35 - 65
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: sterile water
- Justification for choice of solvent/vehicle: relative non-toxicity for the animals
- Amount of vehicle: All animals received a single standard volume orally. A correction factor of 1.26 was applied based on preliminary data of the sponsor.
- Lot/batch no.: 113318061

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in sterile water.

Duration of treatment / exposure:

24 h for all dose groups and in addition 48 h for additional animals of vehicle control and 2000 mg/kg bw group

Frequency of treatment:

1 application

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

2 groups of animals were treated with this dose for collection after 24 and 48 h.

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 in test groups, 5 in control groups

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 40 mg/kg bw, Volume of 10 ml/kg bw (in sterile water)
- Lot/batch no.: A0302605

Examinations

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 48 hours. Three adequately spaced dose levels spaced by a factor of 2 were administered.

TREATMENT AND SAMPLING TIMES: Sampling of the bone marrow was done 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then 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:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

A test item was classified as mutagenic if it induced 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 failed to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes was considered non-mutagenic in this system.

Statistics:

nonparametric Mann-Whitney test

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose: The animals treated with 2000 mg/kg bw showed no clinical signs.
- Clinical signs of toxicity in test animals: no
- Other: No sex specific differences were observed with regard to clinical signs. In accordance with the test guidelines the main study was performed using males only.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. In fact the mean values of micronuclei observed after treatment with the test substance were below the value of the vehicle control group.
- Statistical evaluation: Statistical analysis was performed only for the positive control (p = 0.004).
- Clinical signs of toxicity in test animals: Ruffled fur was detected in some animals of the 2000 mg/kg bw dose groups. The treated animals in other dose groups did not express any clinical signs.

Any other information on results incl. tables

Table 1: Summray of the results

Test group	Dose [mg/kg bw]	Sampling time [h]	PCEs with micronuclei [%]	Range	PCE per 2000 erythrocytes
Vehicle	0	24	0.15	1-6	1441
Test item	500	24	0.143	1-7	1502
Test item	1000	24	0.136	1-4	1377
Test item	2000	24	0.186	0-5	1335
Positive control	40	24	1.58	22-56	1437
Vehicle	0	48	0.19	1-7	1455
Test item	2000	48	0.143	2-5	1387

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

A study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h (vehicle and 2000 mg/kg bw only) after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The dose levels of the test item were investigated based on results of a pre-experiment. Administered doses were 500, 1000 and 2000 mg/kg bw. A correction factor of 1.26 was applied based on test item purity and data provided by the sponsor. After treatment with the test item the mean number of PCEs per 2000 erythrocytes in the high dose group after 24 and 48 hour treatment was slightly lower as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control. However, only few individual values in the high dose group fell minimally below the lowest individual vehicle control value thus indicating that the test item exerts only a minimal cytotoxic effect in the bone marrow, if at all. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.