A mixture of: 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea; 3-cyclohexyl-1-(4-(4-(3-octadecylureido)benzyl)phenyl)urea; 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

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:

21 May 1990 - 22 August 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 May 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay."

Version / remarks:

01 July 1986

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1‐cyclohexyl‐3‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

EC Number:

604-940-8

Cas Number:

154099-21-5

Molecular formula:

C39 H62 N4 O2

IUPAC Name:

1‐cyclohexyl‐3‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Constituent 2

Reference substance name:

3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

EC Number:

406-370-3

EC Name:

3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

Cas Number:

58890-25-8

Molecular formula:

C27 H36 N4 O2

IUPAC Name:

3‐cyclohexyl‐1‐[4‐({4‐[(cyclohexylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Constituent 3

Reference substance name:

3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

EC Number:

406-690-3

EC Name:

3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

Cas Number:

43136-14-7

Molecular formula:

C51 H88 N4 O2

IUPAC Name:

3‐octadecyl‐1‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Test material form:

solid

Details on test material:

Appearance: White yellowish solid
Storage conditions: At room temperature in the dark

The data on the individual constituents CHA/MDI/CHA (EC 406-370-3) and ODA/MDI/ODA (EC 406-690-3) for physicochemical, environmental fate, ecotoxicological and mammalian toxicological endpoints are very similar to the data available for the different compositions of the multi-constituent or represent the outer boundaries of the data on the multi-constituent. Hence, they confirm that the different compositions are expected to have similar properties for physicochemical, environmental fate, ecotoxicological and mammalian toxicological endpoints.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, Füllingsdorf/Basel, Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: 18 h before treatment, water ad libitum
- Housing: Individually housed in Makrolon cages.
- Diet: free access pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Water: free access to tap water
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used:corn oil
- Justification for choice of vehicle: The test substance was suspended in corn oil. The vehicle was chosen for its nontoxicity in animals.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of experiment, the test substance was suspended in corn oil.

MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg bw.

Frequency of treatment:

Single dose.

Post exposure period:

Sampling of the bone marrow was done 24, 48 and 72 h after treatment

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

12 (6 males/6 females)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral (single dose)
- Doses / concentrations: 40 mg/kg bw
- Volume applied: 10 mL/kg bw.
- sampling 24 h after treatment

Examinations

Tissues and cell types examined:

Bone marrow
Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE / normochromatic erythrocytes (NCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose, which was determined to be the dose that caused toxic reactions without having major effects on survival within 72 h.

TREATMENT AND SAMPLING TIMES:
Sampling of the bone marrow was done 24, 48 and 72 h after treatment.

DETAILS OF SLIDE PREPARATION:
Epiphyses were cut off the femore and the marrow was flushed out with fetal calf serum. The cell suspension was centrifuged at 1500 rpm for 5 minutes and the supernatant discarded. A small drop of the resuspended cell pellet was spread on a slide, air-dried and stained with May-Grünwald/Giemsa. Cover slips were mounted wit EUKITT.

METHOD OF ANALYSIS:
Evaluation was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between PCEs and normochromatic erythrocytes (NCEs) was determined in same sample and expressed in the number of NCEs per 1000 PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statitically significant and reproducible positive response at anyone of the test points is considered non-mutagenic in this system.

Statistics:

nonparametric Mann-Whitney test

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Animals expressed slight toxic reactions, no cytotoxicity observed

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- 4 animals (2 males/2 females) received orally a single dose of 5000 mg/kg bw MDI/CHA/ODA.
- All treated animals expressed slight toxic reactions: reduction of spontaneous activity. Additionally two males and one female expressed apathy.
- Higher dosing was not attainable: Appropriate suspensions could be obtained only up to 250 mg/mL and application volumes higher than 20 mL/kg bw were not justifiable for the rodents used.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no significant enhancement in the frequency of the detected micronuclei at 24 h, 48 h or 72 h.
- Ratio of PCE/NCE: no increase after treatment with the test substance indicating that the test substance had no cytotoxic properties.
- Positive control: showed distinct increase of induced micronucleus frequency (mean 12 per 1000 PCE vs. mean 0.4 per 1000 PCE in vehicle control)

Applicant's summary and conclusion

Conclusions:

In a micronucleus assay in the bone marrow of the mouse, performed according to OECD 474 guideline and GLP principles, MDI/CHA/ODA was found not to be mutagenic.

Executive summary:

A micronucleus assay in the bone marrow of male and female mouse with MDI/CHA/ODA was performed according to OECD 474 guideline and GLP principles. Bone marrow cells were collected for micronuclei analysis 24 h, 48 h and 72 h after a single application of 5000 mg/kg bw of the test article. In a pre-experiment, this dose was determined to be the maximum attainable dose. The animals expressed slight toxic reactions (apathy and reduction of spontaneous activity).

After treatment with the test substance the ratio between polychromatic erythrocytes and normochromatic erythrocytes was not affected indicating no cytotoxic effects. There was no significant enhancement in the frequency of detected micronuclei at any preparation interval. Appropriate negative and positive controls were included.

Based on the results of this study it is concluded that MDI/CHA/ODA is not clastogenic in the micronucleus assay in the bone marrow of the mouse.