2,4,6,8-tetramethyl-1,3,5,7-tetraoxacyclooctane

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:

February 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6,8-tetramethyl-1,3,5,7-tetraoxacyclooctane

EC Number:

203-600-2

EC Name:

2,4,6,8-tetramethyl-1,3,5,7-tetraoxacyclooctane

Cas Number:

108-62-3

Molecular formula:

C8H16O4

IUPAC Name:

2,4,6,8-tetramethyl-1,3,5,7-tetraoxocane

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Commercial name/code: P0071
- Appearance: White powder
- Storage: at apprx. 4°C

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm
the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared in-house on 23 March 1998 from the livers of male SpragueDawley rats weighing - 200g. These had each received a single i.p. injection of Aroclor 1254 at 500 mg/kg, five days before 59 preparation.

Test concentrations with justification for top dose:

Pretest:
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 ug/plate; the test material was non-toxic to the strains of bacteria used (TA 100 and WP2uvrA}

Main test:
Based on the results of the pretest, the following does were selected for the main study: 0, 50, 150, 500, 1500, 5000 ug/plate

Vehicle / solvent:

DMSO (Dimethylsulphoxide)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)

DURATION
- Preincubation period: ca. 10h and 48h at 37°C
- Exposure duration: no data
- Expression time (cells in growth medium): no data
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): no data

Rationale for test conditions:

Results of pretest

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

This study was conducted to assess the mutagenic potential of the test material using a bacterial/microsome test system. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia

coli strain WP2 uvrA were treated with suspensions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.