2-Butanone, peroxide

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:

2009-11-27 to 2009-11-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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-[(2-hydroperoxybutan-2-yl)peroxy]butane-2-peroxol; butane-2,2-diperoxol

EC Number:

700-954-4

Cas Number:

1338-23-4

Molecular formula:

Mixture of C4H10O4 and C8H18O6

IUPAC Name:

2-[(2-hydroperoxybutan-2-yl)peroxy]butane-2-peroxol; butane-2,2-diperoxol

Method

Target gene:

Bacterial strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used.
His-mutations D6610 (TA97a) and D3052 (TA98) are frameshift mutations, reversion can occur by addition or deletion of base-pairs. His-mutation G46 (TA100) is a base pair mutation, reversion occurs by the substitution of a single base. In G428 (his-mutation of TA102) th codon CAA, coding for gutamine is mutated to the stop-codon TAA. The functionality of the wild type can be restored by base pair mutation as well as by deletion of 3 or 6 bases.
The rfa mutation leads to a reduced lipopolysaccharide barrier in the cell wall and allows larger molecules to pass the cell wall. Bacteria with this mutation are sensitive to crystal violet.
UvrB results in a loss of DNA-excision repair system. This increases the sensitivity to mutagenic influences. Bacteria with this mutation are sensitive to UV light.
The plasmid pkM101 also disturbs the ability of the bacteria to repair genetic damage and therefore increases their sensitivity to mutagens. Bacteria with this plasmid are resistant against ampicillin. TA102 is also resistant against tetracycline.

Species / strain

Species / strain / cell type:

other: S. typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

In preliminary toxicity test, the following concentrations of test substance solutions are used: 5000, 1667, 556, 185, 62 and 21 µg/plate.
Main test: 556 µg/plate was chosen as highest concentration which could be in the toxic range, and a total of 6 concentrations was tested.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

2-nitrofluorene

sodium azide

other: 4-Nitro-o-phenylenediamine (TA97, without S9); t-Butyl-hydroperoxide (TA102, without S9); 2-aminoanthracene (TA98, TA100, TA1535, with S9); 1,8-dihydroxy-anthraquinone (TA102, with S9)

Details on test system and experimental conditions:

One day before the Ames test was performed, a small amount from each of the frozen bacterial cultures was transferred to nutrient. The liquid cultures were incubated in a shaker overnight at 37 °C and then used for the exposure. The mean number of viable cells in the overnight-cltures is 2 to 3 x10e9 cells per mL, based on historical data.

Evaluation criteria:

Calculations, criteria for a positive result:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2 1/2 fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1 2/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of historic data of the Ames test.

Statistics:

Means and standard deviations were calculated for the number of mutants in every concentration group.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the main test, the test substance was again toxic to bacteria at 556 µg/plate and some of the 185 µg/plate samples. At 62 µg/plate and beneath the bacterial background was normal. Only strain TA102 was not affected.
There was no such increase in the number of mutants in any of the tested bacterial strains at any of tested concentrations. The addition of an external metabolising system did not change these results.

Applicant's summary and conclusion

Conclusions:

According to these results, methyl-ethylketone peroxide in TXIB/diacetone alcohol is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 556 µg/plate, which is the limit of toxicity.

Executive summary:

Methyl-ethylketone peroxide in TXIB/diacetone alcohol was tested in the Salmonella typhimurium reverse mutation test (Ames test) according to OECD guideline 471 and EU method B.13/14. Five bacterial strains, Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used to investigate the mutagenic potential of methyl-ethylketone peroxide in two independent experiments. These experiments were carried out with and without metabolic activation (S9 mix). Triplicate repetitions were run for each dose group in each of two seperate experiments that were conducted, for the control groups six-fold repetitions were run. The exposure for the first experiment was performed according to the Plate Incorporation Assay. The exposure for the second experiment was performed according to the Preincubation Assay. All positive and negative (solvent) control groups were in the range of the historical control range and demonstrated the sensitivity and validity of the test. There was no biologically relevant increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change the results.

Methyl-ethylketone peroxide is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 556 ug/plate, which is the limit of toxicity.