2-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-documented experiment according to GLP and EC and OECD guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane

EC Number:

408-200-3

EC Name:

2-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane

Cas Number:

63187-91-7

Molecular formula:

C13H24O3

IUPAC Name:

[9-methyl-6-(propan-2-yl)-1,4-dioxaspiro[4.5]decan-2-yl]methanol

Test material form:

other: liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

1.0; 10.0; 33.3; 100.0; 333.3; 1000.0 and 5000.0 µg/plate

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine (4-NOPD) and 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

Details on strains used:
- TA 1537: his C 3076; rfa-; uvrB-; frame shift mutations
- TA 1538: his D 3052; rfa-; uvrB-; frame shift mutations
- TA 98: his D 3052; rfa-; uvrB-; R-factor; frame shift mutations
- TA 1535 his G 46; rfa-; uvrB-; base-pair substitutions
- TA 100 his G 46; rfa-; uvrB-; R-factor; base-pair substitutions


Details on experimental performance:
For each strain and dose level, including the controls, a minimum of 3 plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100µL test solution at each dose level, solvent control, negative control or reference mutagen solution
- 500 µL S9 mix (for tests with metabolic activation) or S9 mix substitution-buffer (for tests without metabolic activation)
- 100 µL bacteria suspension (pre-culture)
- 2000 µL overlay agar

Data recording:
The colonies were counted using the BIOTRAN III counter.

Evaluation criteria:

Evaluation criteria:
A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant 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 of the test points is considered non-mutagenic in this sytem.

A significant response is described as follows:
A test article is considered as mutagenic if in strains 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:

Not performed.

Results and discussion

Test results

Test results 1

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

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test article did not induce point mutations by base pair changes or frame shifts in the genome of the strains used.

Executive summary:

The study investigates the potential of the test substance to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The study was performed according to GLP and OECD and EU guidelines. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 1.0, 10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate.

 

Distinct toxic effects, evidenced by a partial or complete reduction in the number of revertants, occurred at higher dose levels in experiment I and II with and without metabolic activation in nearly all strains used. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used, except in experiment II, where the background growth was reduced in the strains TA 1537 and TA 1538 at 5000 µg/plate with and without metabolic activation.

 

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

 

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frame shifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.