A mixture (1:2:1) of: bis(N-cyclohexyl-N'-phenyleneureido)methylene; bis(N-octadecyl-N'-phenyleneureido)methylene; bis(N-dicyclohexyl-N'-phenyleneureido)methylene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

april 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC directive 84/449, L 251, B14

Version / remarks:

p. 143-145

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

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

Method

Target gene:

- S. typhimurium: Histidine gene

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 100, TA 1537, TA 1538 and TA 98

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and TA98: 1.0, 3.3, 10.0, 33.3, 100.0, 333.3, 1000, and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 10.0, 100.0, 333.3, 1000, and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 10.0, 100.0, 333.3, 1000, and 5000 µg/plate

Vehicle / solvent:

- solvent used: DMSO
- Justification for choice of solvent: A homogeneous suspension could be in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 10 µg/plate in aquadest for TA1535 and TA100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

without S9 50 µg/plate in DMSO for TA1537, TA1538 and TA98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

with S9 10 µg/plate in DMSO for all tester strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction in the number of spontaneous revertants, the clearing of the bacterial background lawn, or by the degree of survival of treated cultures.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a siginificant and reproducible increase for at least one test concentration is induced.

A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method is available

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- The test article precipitated weakly from 333.3 µg/plate up to and including the highest concentration in the overlay agar. The undisolved particles of the test article had no influence on the data recording.

RANGE-FINDING/SCREENING STUDy:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate except for strain TA 1535, in which a reduction in the number of revertants was observed at 5000 µg/plate with and without S9.

Applicant's summary and conclusion

Conclusions:

In a Salmonella typhimurium reverse mutation assay, performed according to OECD guideline and GLP principles, MDI/CHA/ODA/DCHA was found not to be mutagenic with or without metabolic activation.

Executive summary:

A Salmonella typhimurium reverse mutation assay was performed according to OECD guideline and GLP principles. All bacterial strains showed negative responses up to and including 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity of the test substance was observed, except for strain TA 1535 for which a reduction in the number of revertants was observed at 5000 ug/plate with and without S9. Negative controls, solvent controls and positive controls (approriate reference mutagens) were included and showed expected results indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that MDI/CHA/ODA/DCHA is not mutagenic in the Salmonella typhimurium reverse mutation assay with or without metabolic activation.