3,5,5-trimethylhexanal

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,5,5-trimethylhexanal

EC Number:

226-603-0

EC Name:

3,5,5-trimethylhexanal

Cas Number:

5435-64-3

Molecular formula:

C9H18O

IUPAC Name:

3,5,5-trimethylhexanal

Details on test material:

Date of production: February/March 1996
Properties: colourless, liquid, homogenous
Stability: > 1 year
ph: not determinate
Purity: 91,2 mass- % (GC analysis)

Method

Target gene:

Four Salmonella typhimurium strains (TA 98, TA 100, TA 1535 and TA 1537) were obtained directly from Dr. Ames, University of Berkeley, USA. They contain specific types of mutations as well-defined locations in different genes of the histidine operon. The strains detec mutations that restore the ability to synthesize histidine, resulting in the growth of visible bacterial colonies on histidine-deficient media.

Test concentrations with justification for top dose:

5 test compound concentrations, both with and without the addition of a metabolising system (Aroclor 1254 induced rat liver S9 mix) were employed
Positive control (with metabolic activation) - 2-aminoanthracene, vehicle DMSO, 2.5 µg/plate
Solvent control, vehicle DMSO, 100 (plate incorporatin) or 50 µl/plate (preincubation test)

Vehicle / solvent:

The tests with a metabolising system were carried out with an Aroclor 1254 induced rat liver S9 fraction.
The enzymatic activity of which was checked on all strains with 2-aminoanthracene.

Details on test system and experimental conditions:

The tester strains were stored as frozen permanents at - 80 C. Fresh working cultures were prepared for each test day by inoculation bacteria from
frozen working cultures into nutrient broth. Cultures were incubated overnight (approx. 18 hrs) at 37 °C with gentle agitation. The bacterial titer was determined by platin a 10 6-dilution of the overnight culture on nutrient agar plates and counting the resultant colonies after 48 hrs incubation at
37 ° C.

Evaluation criteria:

For a test compound to be considered positive, it must (in two independent experiments) cause at lest a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test
article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-
related and not reproducible in two independent tests, this will be taken as an indication of a mutgenic effect.

Statistics:

Bacterial colonies were counted on an Artek Model 880 Colony Counter which was calibrated for each test to check the counting accuracy. In
addition, an examination of the bacterial background lawn on each plate was made. If no background lawn was observed, this was recorded and
no plate count was performed. If the background lawn was reduced as compared to the solvent control, this was also recorded, but colonies were counted. A reduction in the number of spontaneous revertants is also indicative of toxicity.
For all replicate platings, the mean number of revertants per plate and the standard deviation around the mean were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Test results 2

Species / strain:

S. typhimurium TA 1537

Remarks:

preincubation and plate incorporation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

15.1W

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Remarks:

preincubation and plate incorporation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Remarks:

preincubation and plate incorporation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Remarks:

preincubation and plate incorporation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

15.1W

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All four bacterial strains exhibited mutagenic responses to the appropriate positive control substance. Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in an acceptable range.
In each test, 5 test compound concentrations, both with and without the addition of a metabolising system (Aroclor 1254 induced rat liver S9 mix) were employed. Limited by the bacteriotoxic activity of the test compound, these concentrations were different for the individual tester strains but did not exceed 5000 µg/plate.

A reproducible mutagenic activity of the test compound to one or more of the tester strains was not observed with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

TRIMETHYLHEXANAL did not include a mutagenic effect in S. typhimurium. It is therefore not considered to be a bacterial mutagen.

Executive summary:

TRIMETHYLHEXANAL was tested for its ability to induce reverse mutations in an in vitro bacterial system.

Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were treated with the test compound by the Ames test plate incorporation (tests #15.1 and #15.1 W) as well as the preincubation method (tests #15.2 and #15.2). In each test, 5 test compound concentrations, both with and without the addition of a metabolising system (Aroclor 1254 induced rat liver S 9 mix) were employed. Limited by the bacteriotoxic activity of the test compound, these concentrations were different for the individual tester strains but did not exceed 5000 µg/plate.

All four bacterial strains exhibited mutagenic responses to the appropriate positive control substances. Solvent controls were also tested with each strain and the mean of numbers of spontaneous revertants were in an acceptable range.

A reproducible mutagenic activity of the test compound to one or more of the tester strains was not observed with and without metabolic activation.