6-(2,4,4-trimethylcyclopentylidene)hexanal

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 13 to 27 May, 2013

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 major deviation but the isomers ratio is not reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

Directive 2000/32/EC

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.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(6E)-6-(2,4,4-trimethylcyclopentylidene)hexanal

Cas Number:

1429808-42-3

Molecular formula:

C14 H24 O

IUPAC Name:

(6E)-6-(2,4,4-trimethylcyclopentylidene)hexanal

Constituent 2

Reference substance name:

(6Z)-6-(2,4,4-trimethylcyclopentylidene)hexanal

Cas Number:

1429808-41-2

Molecular formula:

C14 H24 O

IUPAC Name:

(6Z)-6-(2,4,4-trimethylcyclopentylidene)hexanal

Test material form:

liquid

Details on test material:

- Physical state: colourless to light yellow translucent liquid
- Storage condition of test material: fridge 6°C±3°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):

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:

Cytotoxicity test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 100 without S9 under the direct plate incorporation method.
Mutagenicity test:
Main test: 0.06, 0.19, 0.56, 1.67 and 5 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: 0.06, 0.19, 0.56, 1.67 and 5 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%)
- Justification for choice of solvent/vehicle: The solubility of the test item was evaluated in a standard solvent panel (milliQ water, ethanol 95%, DMSO and corn oil). Observation of precipitation by the unaided eye indicated that the test item is not soluble. The test item was found to be soluble when diluted 1/3 in ethanol (96%).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

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:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TESTER STRAINS: 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 48 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 48 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
- Sterility test: the sterility of the test item was assayed by adding of 5μL/plate to a minimal agar plate and incubating at 37ºC for 48h.
- Solubility test: the solubility of the test item was evaluated in a standard solvent panel (milli Ro 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 found to be soluble when diluted 1/3 in ethanol (96%)

Evaluation criteria:

The criteria used for determining a positive result take into account a dose-response effect 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.
A result is considered positive whenever the number of revertants of the test item treated plates is increased when compared to the solvent treated plates according to the following criteria: 2 fold for S.typhimurium TA98, TA 100 and E.coli WP2(pKM101); 3 fold for S.typhimurium TA1535 and TA1537.
Biological relevance of the results was also considered.

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:

cytotoxicity

Remarks:

at 5 μL/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was found to be soluble when diluted 1/3 in ethanol (96%).
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST:
A decrease in the number of revertant colonies >50% compared to solvent reference was observed, indicating cytotoxicity of the test item. The lowest cytotoxic oncentration was used as the highest formulation (5 μL/plate).

MUTAGENICITY TEST:
- None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure.
- No dose response for the test item 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, test item is not mutagenic with and without 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 substance diluted in ethanol (96%) at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

 

Cytotoxicity test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 100 without S9 under the direct plate incorporation method. 

Mutagenicity test:

Main test: 0.06, 0.19, 0.56, 1.67 and 5 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: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 (pKM101) with and without S9 under the pre-incubation method.

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

 

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data.

 

A decrease in the number of revertant colonies > 50% compared to solvent reference was observed, indicating cytotoxicity of the test item. The lowest cytotoxic concentration was used as the highest formulation (5 µL/plate). None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure. No dose response for the test item was observed in any of the tested bacterial strains.

 

Under the test condition, test substance 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.