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Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 1990 - 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 26, 1983
Qualifier:
according to guideline
Guideline:
other: EEC DIrective 84/449, L 251, B 12
Version / remarks:
p. 137-139
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:
July 1, 1986
GLP compliance:
yes
Type of assay:
other: micronucleus assay in bone marrow cells of the mouse

Test material

Constituent 1
Reference substance name:
A mixture (1:2:1) of: bis(N-cyclohexyl-N'-phenyleneureido)methylene; bis(N-octadecyl-N'-phenyleneureido)methylene; bis(N-dicyclohexyl-N'-phenyleneureido)methylene
EC Number:
406-550-1
EC Name:
A mixture (1:2:1) of: bis(N-cyclohexyl-N'-phenyleneureido)methylene; bis(N-octadecyl-N'-phenyleneureido)methylene; bis(N-dicyclohexyl-N'-phenyleneureido)methylene
IUPAC Name:
Reaction mass (1:2:1) of bis(N-cyclohexyl-N'-phenyleneureido)methylene and bis(N-octadecyl-N'-phenyleneureido)methylene and bis(N-dicyclohexyl-N'-phenyleneureido)methylene
Test material form:
solid: particulate/powder
Details on test material:
Description: White yellowish solid
Test substance storage: At room temperature in the dark

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: 10 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: single
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

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

IN-LIFE DATES: From: May 21, 1990 To: June 12, 1990

Administration / exposure

Vehicle:
VEHICLE: cornoil
- Justification for choice of vehicle: Vehicle was chosen for its nontoxicity in animals.
Details on exposure:
DOSAGE PREPARATION: On the day of the experiment, the test article

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
- Doses / concentrations: 40 mg/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
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) recieved orally a single dose of 5000 mg/kg bw MDI/CHA/ODA/DCHA.
- All treated animals expressed slight toxic reactions: reduction of spontaneous activity. additionally one male and two females 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 article indicating test article had no cytotoxic properties
- Positive control: showed distinct increase of induced micronucleus frequency (66 per 1000 PCE vs. 8 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 guideline and GLP principles, MDI/CHA/ODA/DCHA was found not to be mutagenic.
Executive summary:

A micronucleus assay in the bone marrow of the mouse with MDI/CHA/ODA/DCHA was performed according to OECD guideline and GLP principles. Bone marrow cells were collected for micronucleui 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 article 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/DCHA is not clastogenic in the micronucleus assay in the bone marrow of the mouse.