1,4-dioxacycloheptadecane-5,17-dione

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:

From 2012-11-01 to 2012-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD test guideline No. 471 and in compliance with GLP

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP (Inspected on 2012-06-20)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-dioxacycloheptadecane-5,17-dione

EC Number:

203-347-8

EC Name:

1,4-dioxacycloheptadecane-5,17-dione

Cas Number:

105-95-3

Molecular formula:

C15H26O4

IUPAC Name:

1,4-dioxacycloheptadecane-5,17-dione

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): Ethylene Brassylate
- Physical state: colourless/pale yellow liquid
- Storage condition of test material: Room temperature, dry.

Method

Target gene:

Histidine gene for S. typhimurium and tryptophan gene for E.coli

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):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (S9 fraction, prepared from male Sprague-Dawley derived rats, dosed i.p. with phenobarbital sodium and 5,6-benzoflavone)

Test concentrations with justification for top dose:

Test 1: 5, 15, 50, 150, 500, 1000 and 5000 µg/plate.
Test 2: 50, 150, 500, 1000 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(See Table 7.2.1/1)

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

In the absence of S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(See Table 7.2.1/1)

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

In the presence of S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1 in agar (plate incorporation), Experiment 2 preincubation

DURATION
- Preincubation period (Exp 2): 30 minutes at 37°C.
- Exposure duration: ca. 72 hours at 37°C.

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: growth assessment of the bacterial background lawn

OTHER: ACCEPTANCE CRITERIA:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10E09/mL.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.

Statistics:

The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weak toxicity at 5000 µg/plate without S-9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: the test material is not classified as a VOC.
- Water solubility: the test material was solubilised in DMSO to improve solubility.
- Precipitation: none
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- First test:
Evidence of weak toxicity, observed as a slight reduction in revertant colony numbers, was obtained in the Salmonella strains following exposure to Ethylene Brassylate at 5000 µg/plate in the absence of S9 mix. No evidence of toxicity was obtained in the E. coli strain, or in the presence of S9 mix. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
- Second test:
Evidence of weak toxicity, observed as a slight reduction in revertant colony numbers, was obtained in the Salmonella strains following exposure to Ethylene Brassylate at 5000 µg/plate in the absence of S9 mix. No evidence of toxicity was obtained in the E. coli strain, or in the presence of S9 mix.

OTHER:
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.
The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 109/mL in all cases, and therefore met the acceptance criteria.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Ethylene Brassylate is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA98, TA100, and E.coli WP2 uvrA according to the criteria of the Annex VI of the of the Regulation (EC) No 1272/2008 (CLP) and of the Directive 67/548/EEC.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA were exposed to Ethylene Brassylate diluted in DMSO both in the presence and absence of metabolic activation system (10% v/v S9 fraction in standard co-factors) using both the Ames plate incorporation and pre-incubation methods, in triplicate. Concentrations of Ethylene Brassylate up to, and including, 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Evidence of weak toxicity, observed as a slight reduction in revertant colony numbers, was obtained in the Salmonella strains following exposure to the test material at 5000 µg/plate in the absence of S9 mix. No evidence of toxicity was obtained in the E. coli strain, or in the presence of S9 mix.

No evidence of mutagenic activity was seen at any concentration of Ethylene Brassylate in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

Under the test condition, Ethylene Brassylate is not mutagenic in the presence and absence of metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.