Reaction mass of 2-ethylhexyl (6-isocyanatohexyl)-carbamate and bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

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:

July-Aug 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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

Reference substance name:

2-ethylhexyl (6-isocyanatohexyl)-carbamate

EC Number:

247-735-5

EC Name:

2-ethylhexyl (6-isocyanatohexyl)-carbamate

Cas Number:

26488-60-8

Molecular formula:

C16H30N2O3

IUPAC Name:

6-Isocyanatohexylamino 3-ethylheptanoate

Constituent 2

Reference substance name:

Bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

EC Number:

278-583-8

EC Name:

Bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

Cas Number:

76977-79-2

Molecular formula:

C24H48N2O4

IUPAC Name:

6-(2-Ethylhexyloxycarbonylamino)hexylamino 3-ethylheptanoate

impurity 1

Reference substance name:

Hexamethylene diisocyanate

EC Number:

212-485-8

EC Name:

Hexamethylene diisocyanate

Cas Number:

822-06-0

Molecular formula:

C8H12N2O2

IUPAC Name:

1,6-diisocyanatohexane

Test material form:

liquid

Specific details on test material used for the study:

- Stability under test conditions: A stability test in the formulation at 0.1 and 100 mg/mL revealed no significant degradation of the test item up to at least 4 hours (A 01/0207/02 LEV).

Method

Target gene:

Histidine-deficient mutant  strains of Salmonella typhimurium LT2

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 the liver of Aroclor 1254 induced rats

Test concentrations with justification for top dose:

Pre-test for cytotoxicity: doses up to 5000 µg/plate (recommended maximum test concentration)
Initial test (plate incorporation): due to bacteriotoxic effects doses up to 128 µg/plate were used (4, 8, 16, 32, 64, and 128 µg/mL)
Independent repeat (pre-incubation method): doses up to 64 µg/tube were used (1, 2, 4, 8, 16, 32, and 64 µg/mL)

Vehicle / solvent:

Ethylene glycol dimethylether (EGDE; dried with molecular sieve)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

EGDE

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other:

Remarks:

Na-azide, NF, 4-NPDA, MMC, and Cumene are only used without S9 mix, 2-AA is only used with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A pre-test for cytotoxicity and the initial mutagenicity test were performed as plate incorporation test, the independent repeat as pre-incubation modification (Pre-incubation time 20 minutes at 37 °C). Each strain and concentration was tested in triplicate.

DETERMINATION OF CYTOTOXICITY:
1. gross appraisal of background growth, 2. mutant count per plate

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 and TA98 this increase should be about twice that of negative controls, whereas for TA1537 at least a threefold increase should be reached. For TA102 an increase of about 100 mutants should be reached.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA98, and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses up to and including 4 µg per plate did not cause any bacteriotoxic  effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 128 µg per plate for assessment purposes.

Applicant's summary and conclusion

Executive summary:

A bacterial reverse mutation test (Ames) equivalent to OECD TG 471 was conducted for the evaluation of point mutagenic effects. In this assay five histidine-deficient mutant strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA 98, and TA 102) were used, with and without a metabolic activating system. The study was performed as initial plate incorporation with the pre-incubation modification as independent repeat. Doses ranged from 1 to 5000 μg/plate.

Doses up to and including 4 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 128 µg/plate for assessment purposes.

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls, and by thus showed the sensitivity of the test system and the activity of the metabolic activating system.

Therefore, the test substance was considered to be non-mutagenic without or with a metabolic activating system in bacteria.