Myrac Aldehyde

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:

13 August 2013 - 05 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines

Version / remarks:

31 March 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

915-650-9

Molecular formula:

C13H20O

IUPAC Name:

Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Test material form:

liquid

Details on test material:

Batch No. VE0027614
Purity: 99.6% (sum of the two main constituents)
Name of the test item (as cited in the study report): MYRALDENE
Physical state: colourless to pale yellow liquid
Storage Conditions: +2°C to +8°C, protected from light
Expiry Date: 12 April 2014

Method

Target gene:

Salmonella typhimurium: histidine gene (his- to his+ reversions)
Escherichia coli: tryptophan gene (trp- to trp+ reversions)

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment/Experiment 1:
- All strains, with and without S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment 2:
Without S9 mix
- Strains TA 1535 and WP2 uvrA: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
- Strains TA 1537, TA 98, TA 100: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate
With S9 mix
- Strains TA 1537 and WP2 uvrA: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
- Strains TA 1535, TA 98, TA 100: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

(untreated plates)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Remarks:

see section ''Any other information on materials and methods incl. tables''

Details on test system and experimental conditions:

A pre-experiment was performed to evaluate the toxicity of the test item. The pre-experiment was performed under the same conditions as the final experiment, using all strains, and was reported as main experiment 1. The main study was performed in two experiments: experiment 1 was a plate incorporation assay, experiment 2 was a pre-incubation assay.

METHOD OF APPLICATION: in agar

DURATION (Pre-incubation assay)
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates for each straind and dose level

DATA RECORDING
Colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager (v.1.21). Due to precipitation of the test item and reduced background growth the 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.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- 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. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 333-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 100-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 100-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 33-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 333-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 2500 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment II at 5000 μg/plate in strains TA 1535 and WP2 uvrA without S9 mix and in strains TA 1537 and WP2 uvrA with S9 mix. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The results of the pre-experiment were reported as experiment 1.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see table 1.

Any other information on results incl. tables

Table 1 Historical data

Strain		Without S9 mix				With S9 mix
		Mean	SD	Min	Max		Mean	SD	Min	Max
TA 1535	Solvent control	14	2.37	9	23		20	3.75	11	35
	Untreated control	14	2.87	7	25		20	3.82	10	32
	Positive control	1751	226.44	710	2385		367	93.12	126	703
TA 1537	Solvent control	12	3.31	5	26		16	4.34	7	30
	Untreated control	12	4.03	5	29		18	4.92	6	33
	Positive control	88	38.92	61	448		342	144.31	77	809
TA 98	Solvent control	30	5.60	17	47		40	6.08	21	58
	Untreated control	31	6.36	17	55		42	6.83	24	68
	Positive control	372	78.05	158	595		2167	717.60	249	4089
TA 100	Solvent control	142	29.42	86	243		156	29.4	99	249
	Untreated control	150	28.19	86	248		163	31.26	94	281
	Positive control	1741	488.75	569	3082		2642	769.59	825	4503
WP2uvrA	Solvent control	49	7.67	32	69		60	8.55	31	86
	Untreated control	50	7.64	31	71		60	8.06	38	83
	Positive control	697	336.39	174	1435		368	108.91	176	1534

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay performed according to OECD 471.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in all strains (>=250 μg/plate). Adequate negative, vehicle and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in neither each of the four S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) nor in the E. coli tester strain (WP2 uvrA), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.