3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers reaction product with 3-(cyclohexylamino)propane-1-sulfonic acid and N,N-dimethylcyclohexanamine

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 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid - solid: mixture of

Specific details on test material used for the study:

- Stability under test conditions: The stability of the substance in the formulation was analytically verified for at least 24 hours.
- The substance in the test item is dissolved in approx. 30 % solvent. Test concentrations in the Ames-test were adjusted to the substance content (approx. 70 %).

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix from rat liver induced with phenobarbital/beta-naphthoflavone.

Test concentrations with justification for top dose:

Experiment I (plate incorporation), with and without S9-mix: 10, 25, 50, 160, 500, 1600, and 5000 µg/plate for each tester strain
Experiment II (pre-incubation method), with and without S9-mix: 10, 25, 50, 160, 500, 1600, and 5000 µg/plate for each tester strain

Vehicle / solvent:

EGDE (Ethylene glycol dimethylether)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

EGDE

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

cumene hydroperoxide

mitomycin C

other: 4-Nitro-1,2-phenylene diamine (used for TA1537), Anthracene-2-amine (used for all strains).

Remarks:

The positive control Anthracene-2-amine was only used with S9 mix, the others were used without S9-mix..

Details on test system and experimental conditions:

METHOD OF APPLICATION: Initially the plate incorporation method was used. As independent repeat the preincubation modification (20 min. at 37°C) was performed.
For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
The toxicity of the substance was assessed by a gross appraisal of background growth on the plates for mutant determination. Moreover, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the solvent controls.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100, TA1537 and TA98 this increase should be about twice that of solvent controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Remarks:

plate incorporation trial

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:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Remarks:

plate incorporation trial

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:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Remarks:

plate incorporation trial

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:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Remarks:

plate incorporation trial

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:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Remarks:

plate incorporation trial

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:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1535

Remarks:

preincubation trial

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/tube

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Remarks:

preincubation trial

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:

not applicable

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 1537

Remarks:

preincubation trial

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/tube for trials without S9-mix, no cytotoxicity for trials with S9-mix, but tested up to limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 98

Remarks:

preincubation trial

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:

not applicable

Positive controls validity:

valid

Test results 10

Species / strain:

S. typhimurium TA 102

Remarks:

preincubation trial

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1600 µg/tube for trials without S9-mix, no cytotoxicity for trials with S9-mix, but tested up to limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the plate incorporation test. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials. The positive controls increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Substance precipitation occurred at 5000 µg per plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses of up to and including 5000 µg per plate did not produce bacteriotoxic effects in the plate incorporation method. The preincubation method showed the test item to produce strain-specific bacteriotoxic effects at the dose of 5000 µg per plate.

Applicant's summary and conclusion

Executive summary:

A bacterial reverse mutation test (Ames test) was conducted according to OECD TG 471 to investigate the potential of the test substance to induce gene mutations. The assay was performed in two independent experiments, the initial plate incorporation and the subsequent preincubation test, both with and without S9 mix and the following tester strains: Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98, and TA 102. The doses applied were up to and including 5000 µg/plate (the content of the substance in the test item, which is approx. 70 % in approx. 30 % solvent, was considered).

Substance precipitation occurred at 5000 µg per plate. None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the plate incorporation test. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

The positive controls increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

In conclusion, the test substance was considered to be non-mutagenic in the Ames test with and without S9 mix.