1,6-Bis(methoxybenzoyloxy)hexane

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:

30 October 2001 - 30 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study includes data generated according to generally valid and internationally accepted testing guidelines and performed according to GLP. The study was based on the following guidelines: -Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (Adopted July 21, 1997). - European Economic Community (EEC). Adapting to technical progress for the twenty-sixth time Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.13/14: "Muta1~enicity: "Reverse Mutation Assay using bacteria". EEC Publication Commission Directive {Published June 8, 2000).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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

Remarks:

GLP Statement date: 10/12/2001 and 21/12/2001

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,6-Bis(methoxybenzoyloxy)hexane

Cas Number:

390359-18-9

Molecular formula:

C22H26O6

IUPAC Name:

1,6-Bis(methoxybenzoyloxy)hexane

Test material form:

other: Yellowish solid

Details on test material:

- Name of test material (as cited in study report): SETAFIX X 11342
- Substance type: Organic
- Physical state: Solid
- Analytical purity: +/- 95%
- Lot/batch No.: PP02
- Expiration date of the lot/batch: 01 July2002
- Stability under test conditions: Not indicated
- Storage condition of test material: Stable

Method

Target gene:

Salmonella typhimurium bacteria and Escherichia coli bacteria

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coIi WP2uvrA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

DOSE RANGE FINDING TEST:
3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of 59-mix.

MUTATION ASSAY:

EXPERIMENT 1: 3, 10, 33, 100, 333 μg/plate in the absence and presence of 59-mix (Strains: TA 1535, TA 1537 and TA98)

EXPERIMENT 2: 3, 10, 33, 100, 333 μg/plate in the absence and presence of 59-mix (Strains: TA 1535, TA 1537, TA98, TA 100 and WP2uvrA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO

- Justification for choice of solvent/vehicle:
Not specified but likely to be solubility of test substance

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strain TA1535

Positive control substance:

sodium azide

Remarks:

Without metabolic activation(-S9-mix)

Controls 2

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation(-S9-mix)

Controls 3

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strain TA98

Positive control substance:

other: Daunomycine

Remarks:

Without metabolic activation(-S9-mix)

Controls 4

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strain TA100

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation(-S9-mix)

Controls 5

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Strain WP2uvrA

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation(-S9-mix)

Controls 6

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strains: TA1537, TA1535, TA98, TA100 and WP2uvrA

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation (+S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Preparation of Bacterial cultures:
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml}. Freshly grown cultures of each strain were used for a test.

Permeabilization of the Escherichia coli strain:
WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-HCL buffer pH 8.0, then gently resuspended in 0.2 vol. 0.12 M TrisHCL, 0.5 mM EDTA pH 8.0, and shaken for 2.5 min at 37°C. MgCl2 was then added to a final concentration of 10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.

Agar Plates:
Agar plates (0 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18g purified agar (Oxoid, code L2) in Vogel-Bonner Medium E, 20 g glucose.
N.B. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan.

Top Agar:
Top agar medium, containing 0.6% (w/v) purified agar and 0.5% (w/v) NaCl, was heated to dissolve the agar. Samples of 3 ml top agar were transferredinto 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 1 °C.

Environmental conditions:
All incubations were carried out in the dark at 37 ± 1 °C. The temperature was monitored during the experiment.

Evaluation criteria:

A test substance is consideried negative (not mutagenic) in the test if:

a) The total number of reveirtants 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 a number of revertant colonies, dose related, greater than two-times the number of revertants 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:

No formal hypothesis testing was done.

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coIi WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

DOSE RANGE FINDING TEST

SETAFIX X 11342 was testeid in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of 59-mix.

Precipitate:

The test substance precipitated in the top agar at concentrations of 100 μg/plate and upwards. Precipitation of SETAFIX X 11342 on the plates was observed at the start and at the end of the incubation period at concentrations of 333 μg/plate and upwards.

Toxicity:

To determine the toxicity of SETAFIX X 11342, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

MUTATION ASSAY

Based on the results of the close range finding test, SETAFIX X 11342 was tested up to concentrations of 333 μg/plate in the absence and presence of S9-mix in two mutation assays.

The first mutation experiment was performed with the strains TA 1535, TA 1537 and TA98 and the second mutation experiment was performed with the strains TA 1535, TA 1537, TA98, TA 100 and WP2uvrA.

Precipitate:

SETAFIX X 11342 precipitated in the top agar at concentrations of 100 and 333 μg/plate. Precipitation of SETAFIX X 11342 on the plates was observed at the start and at the end of the incubation period in all tester strains at the highest concentration tested.

Toxicity

The bacterial background lawn was not reduced at all concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Number of revertants:

All 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 control values were within our laboratory background historical control data ranges. The 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, except for TA1537 in the presence of S9 -mix (second experiment). However, since this value was just without the limit of the range, the validity of the test was considered to be not affected.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that SETAFIX X 11342 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

 

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.

 

SETAFIX X 11342 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 dose range finding test, SETAFIX X 11342 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. SETAFIX X 11342 precipita1ted on the plates at dose levels of 333 μg/plate and upwards. The bacterial background lawn was not reduced at all concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

In the first and in the second mutation assay, SETAFIX X 11342 was tested up to concentrations of 333 μg/plate in the absence and presence of S9-mix. SETAFIX X 11342 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

 

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

 

SETAFIX X 11342 did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

Based on the results of this study it is concluded that SETAFIX X 11342 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.