dimethyl 1,2-benzenedicarboxylate;reaction mass of: 1,2-dimethylpropylidene dihydroperoxide

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

Study period:

22 January 2002 - 31 January 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-480-8

EC Name:

-

Cas Number:

182893-11-4

Molecular formula:

Mixture of C5H12O4 and C10H22O6

IUPAC Name:

reaction mass of 1,2-dimethylpropylidene dihydroperoxide and dimethyl 1,2-benzenedicarboxylate

Details on test material:

Identification: Trigonox R-938
Chemical name: Methyl Isopropyl Ketone Peroxide
CASRN: Mixture of 13921-99-8, 33372-83-7, 131-11-3 and 563-80-4
Description: Clear colourless liquid
Batch: 1510-14
Test substance storage: In refrigerator in the dark
Stability under storage conditions: Stable
Expiry date: 01 January 2003

Method

Target gene:

histidine + typtophan operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see below

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: see below

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

1st experiment:
all strains: 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix.
2nd experiment:
TA1535, TA1537, TA98 and TA100
With and without S9-mix: 10, 33, 100, 333 and 1000 µg/plate
WP2uvrA
Without S9-mix: 10, 33, 100, 333, 1000 and 2000 µg/plate.
With S9-mix : 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: NA
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine/tryptophan

NUMBER OF REPLICATIONS: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: The revertant colonies (histidine independentc c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present.

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision

Statistics:

None

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see results table

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see results table

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see results table

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see results table

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: Not examined

RANGE-FINDING/SCREENING STUDIES: Combined with first experiment

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Any other information on results incl. tables

In the second experiment in tester strain TA1537, MIPKP induced an up to 2.5-fold increase in the absence of S9-mix. However, this increase was only observed in one experiment and the highest number of revertants was not higher than 20 and within our historical control data range. Therefore, this increase is considered to be not biologically relevant and the test substance is considered to be not mutagenic. All other bacterial strains showed negative responses over the entire dose range, i.e. no dose related, two-fold, increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the combined range finding test/first mutation assay, MIKP was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. MIPK did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains. In the second mutation assay, it was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in the strains TA1535, TA1537, TA98 and TA100. TRIGONOX R-938 was tested up to concentrations of 2000 and 1000 µg/plate in the absence and presence of S9-mix, respectively in strain WP2uvrA. Toxicity was observed in all tester strains. The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

In the second experiment in tester strain TA1537, MIKP induced an up to 2.5-fold increase in the absence of S9-mix. However, this increase was only observed in one experiment and the highest number of revertants was not higher than 20 and within our historical control data range. Therefore, this increase is considered to be not biologically relevant and the test substance is considered to be not mutagenic.

All other bacterial strains showed negative responses over the entire dose range, i.e. no doserelated, two-fold, increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that MIPKP is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.