1-Cyclohexene-1-propanal, 4-(1-methylethyl)-, (4R)-

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:

weight of evidence

Study period:

2012-10-23 to 2012-12-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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:

adopted 1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-[(4R)-4-(propan-2-yl)cyclohex-1-en-1-yl]propanal

EC Number:

700-886-5

Cas Number:

1378867-81-2

Molecular formula:

C12H20O

IUPAC Name:

3-[(4R)-4-(propan-2-yl)cyclohex-1-en-1-yl]propanal

Method

Target gene:

not applicable

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital and by peroral administrations of β-naphthoflavone each, on three consecutive days.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (all strains; with and without metabolic activation)
Experiment Ia and II: 0.1; 0.3; 1; 3; 10; 33; 100; and 333 µg/plate (Salmonella strains; without metabolic activation)
Experiment II: 0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate (Salmonella strains; with metabolic activation)
Experiment II: 3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate (E. coli strain; with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (first experiment:MERCK, 64293 Darmstadt/Germany; purity > 99 %; all further experiments: Acros, New Jersey, USA, purity > 99 %).
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria (Maron et al., 1981)*.

*Reference:
- Maron D.M., J. Katzenellenbogen, and B.N. Ames (1981) Compatibility of organic solvents with the Salmonella/Microsome Test Mutation Res. 88, 343-350

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control without metabolic activation: dissolved in water; concentration: 10 µg/plate; strains: TA1535 and TA100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Positive control without metabolic activation: dissolved in DMSO; concentration: 10 µg/plate (strain TA98) and 50 µg/plate (strain TA1537); strains: TA98 and TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control without metabolic activation: dissolved in deionised water; concentration: 3 µL/plate; strain: WP2 uvrA

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control with metabolic activation: dissolved in DMSO; concentration: 2.5 µg/plate (strains TA1535, TA1537, TA98, TA100) and 10 µg/plate (strain WP2 uvrA); strains: TA1535, TA1537, TA98, TA100 and WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (experiment I and Ia) and preincubation (experiment II)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours at 37°C

NUMBER OF REPLICATIONS: for each strain and dose level including the controls, three plates were used.

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. Due to precipitation of the test item and reduced background growth the revertant colonies were partly counted manually.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (Hollstein et al., 1979)*.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (de Serres & Shelby, 1979)*.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. Whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

*References:
- de Serres F.J. and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Mutation Res. 64, 159-165
- Hollstein,M., J. McCann, F.A. Angelosanto, and W.W. Nichols (1979) Short-term tests for carcinogens and mutagens. Mutation Res. 65, 133-226

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

CYTOTOXICITY:
- plates incubated with the test item showed reduced background growth with and without metabolic activation in all experiments (please refer to the table 1 in the field "Any other information on results incl. tables" below).
- toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation (please refer to the table 2 in the field "Any other information on results incl. tables" below).

MAIN EXPERIMENTS
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate)

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	33 - 5000 33 – 333 (HV1a)	333 - 5000	33 - 333	333 - 1000
TA 1537	33 - 5000 33 - 333 (HV1a)	333 - 5000	33 - 333	333 - 1000
TA 98	100 - 5000	333 - 5000	33 - 333	333 - 1000
TA 100	33 - 5000	333 - 5000	100 - 333	333 - 1000
WP2 uvrA	5000	1000 - 5000	2500 - 5000	5000

HV1a: Results for experiment Ia. Due to strong toxic effects in strains TA1535 and TA1537 in experiment I without S9 mix, this part was repeated with lower concentrations.

Table 2:

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	100 – 5000 100 – 333 (HV1a)	333 - 5000	33 - 333	333 - 1000
TA 1537	33 – 5000 100 – 333 (HV1a)	333 - 5000	10 - 333	333 - 1000
TA 98	100 - 5000	333 - 5000	33 - 333	333 - 1000
TA 100	100 - 5000	333 - 5000	100 - 333	333 - 1000
WP2 uvrA	2500 - 5000	1000 - 5000	2500 - 5000	2500 - 5000

HV1a: Results for experiment Ia. Due to strong toxic effects in strains TA1535 and TA1537 in experiment I without S9 mix, this part was repeated with lower concentrations.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
According to Directive 67/548 /EEC and its subsequent amendments and according to Regulation (EC) No 1272/2008 and subsequent regulations, the test substance should not be considered to have a mutagenic potential, and hence no classification or labelling is required.