2-benzyl-4-methyl-4a,5,6,7,8,8a-hexahydro-4H-benzo[d][1,3]dioxine

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:

From 14 to 28 November, 2011.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation. The substance is adequately identified, but some data on composition is missing. Therefore validation applies with restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix. No information on the characterisation of S9 with a mutagen that requires metabolic activation by microsomal enzymes has been included in the report.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2011-10-19, 20 &21 and 2011-11-17 / Signed on 2011-11-17.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-benzyl-4-methyl-octahydro-1,3-benzodioxine

EC Number:

956-606-9

Cas Number:

1373821-23-8

Molecular formula:

C16 H22 O2

IUPAC Name:

2-benzyl-4-methyl-octahydro-1,3-benzodioxine

Test material form:

liquid

Details on test material:

- Physical state: colourless liquid.
- Storage condition of test material: fridge 6°C±3°C, dark

Specific details on test material used for the study:

- colour: Colorless to pale yellow

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):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH
- method of preparation of S9 mix : not detailed
- concentration of S9 in the final culture medium: S9-fraction 10% v/v
- quality controls of S9: enzymatic activity, sterility, metabolic capability (certificate included in the study report)

Test concentrations with justification for top dose:

Migrated Data from field(s)
Field "Justification for deviation from the high dose level" (Path: ENDPOINT_STUDY_RECORD.GeneticToxicityVitro.MaterialsAndMethods.Method.JustificationForDeviationFromTheHighDoseLevel): Cytotoxicity test: 0.02, 0.06, 0.19, 0.56 and 1.67 µL/plate in TA 100
Mutagenicity test:
Main test: 0.02, 0.06, 0.19, 0.56, 1.67 µL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.02, 0.06, 0.19, 0.56, 1.67 µL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol 96%
- Appearance in solvent: liquid solution
- Basis for dose calculation: The test item was used as provided
- Formulation conditions: Room temperature
- Formulation frequency: Daily
- Formulation preparation: The highest exposure concentration was 1.67 μL/plate. Further lower concentrations were prepared by 1:3 serial dilutions in the selected solvent from the highest concentration.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol (96%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ehanol (96%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TESTER STRAINS: Strains of salmonella typhimurium and E. Coli were obtained from Moltox. Bacterial strains used for the study were grown from controlled Working Banks obtained from Master Banks (generated in Vivotecnia).

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 37 °C for 72 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.

OTHER:
- After an incubation of about 72 hours at about 37 ºC, the number of colonies per plate was counted.
Data are presented as the number of colonies present per plate (mean ± standard deviation). The R ratio is calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
- Test item solubility: The solubility of the test item was evaluated in a standard solvent panel (milliQ water, ethanol 96%, DMSO and corn oil). Observation of precipitation by the unaided eye indicated that the test item is not soluble. The test item was soluble in ethanol (96%)at a concentration of 50.0mg/mLbut the solution precipitates in the final treatment mixture until the test item concentration of 16.7mg/mL, which was used as the highest concentration for the cytotoxicity assay.
- Test item sterility assay: The sterility of the test item was assayed by adding of 5mg/plate to a minimal agar plate and incubating at 37ºC for 48h. No growth was observed in the minimal agar plate after incubation with the test item.

Evaluation criteria:

Several criteria are used for determining a positive result: a dose-response in the range tested and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Positive results from the bacterial reverse mutation test indicate that a test item induces point mutations or frame-shifts in the genome of the tested bacterial strains.
Negative results from the test indicate that under the test conditions, the test item neither mutagenic nor-pro-mutagenic in the tested experimental system.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST:
No cytotoxic effect was observed.

MUTAGENICITY TEST:
- No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation.
− No dose response was observed in any of the tested bacterial strains.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.

OTHERS:
- Sterility test showed no contamination during the study.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test condition, the test material is not mutagenic in the presence and absence of metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the test item at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

 

Main test: 0.02,0.06, 0.19, 0.56 and 1.67 µL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.

Confirmation test: 0.02, 0.06, 0.19, 0.56 and1.67 µL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.

 

Negative and positive control groups were also included in mutagenicity tests.

 

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. All positive controls showed valid ratios (R) above 2.5.

 

No cytotoxic effect was observed. No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation. No dose response was observed in any of the tested bacterial strains.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

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