Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Justification for type of information:

Data is from study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

This study was performed to investigate the potential of the test chemical to induce gene mutation according to Ames metabolic activation test.

GLP compliance:

not specified

Type of assay:

other: Bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(3E)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)but-3-en-2-one; (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one

EC Number:

907-706-6

Molecular formula:

C13H20O

IUPAC Name:

(3E)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)but-3-en-2-one; (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one

Test material form:

liquid

Details on test material:

- Name of test material: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- IUPAC name: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- Molecular formula: C13H20O
- Molecular weight: 192.3 g/mole
- Smiles : C1([C@@H](C(=CCC1)C)\C=C\C(C)=O)(C)C
- Inchl: 1S/C13H20O/c1-10-6-5-9-13(3,4)12(10)8-7-11(2)14/h6-8,12H,5,9H2,1-4H3/b8-7+
- Substance type: Organic
- Physical state: Liquid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data available

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction (S-9 mix).

Test concentrations with justification for top dose:

Bacteriostatic test: 10, 1, 0.1, 0.01 µl/well
Mutation test: 1, 0.1, 0.01, 0.001 µl/well

In the Bacteriostatic test Ionone showed toxicity towards the bacteria, therefore a top concentration of 1 µl/plate was chosen for the mutation study.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: 2-amino-anthracene

Controls 3

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Neutral red

Details on test system and experimental conditions:

No data available

Rationale for test conditions:

No data available

Evaluation criteria:

The plates were observed for an increases in the revertant colony numbers of any of the five strains

Statistics:

No data available

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data available

Remarks on result:

other: no mutagenic potential

Any other information on results incl. tables

Table 1

Bacteriostatic test on the test chemical

Strain S. typhimurium	Concentration of Ionone (µl/well)	Zone of inhibition on his^-medium well (mm)
TA 1535	10 1 0.1 0.01 0	28 14 10 10 10
TA 1537	10 1 0.1 0.01 0	Strain failed to grow
TA 1538	10 1 0.1 0.01 0	20 14 10 10 10
TA 98	10 1 0.1 0.01 0	25 17 10 10
TA 100	10 1 0.1 0.01 0	29 12.5 10 10 10

* N.B. well diameter = 10 mm

Table 2

Revertant colony counts obtained per plate using S. typhimurium strains TA 1535, TA 1537 and TA 1538

 

Strain S. typhimurium	Concentration of test material (µl/plate)	Metabolic activation	Mean revertant colony counts	Individual revertant colony counts
TA 1535	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL IL 12 16 13 NL 10 10 16 12	NL IL 13, 10, 12 19, 16, 13 16, 12, 10 NL 9, 7, 14 10, 11, 10 17, 15, 16 12, 10, 14
TA 1537	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL NL 5 6 7 NL IL 8 6 8	NL NL 7, 4, 5 7, 6, 5 7, 7, 6 NL IL 8, 8, 8 6, 7, 6 6, 8, 10
TA 1538	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL IL 16 15 14 NL 13 14 15 14	NL IL 16, 18, 15 13, 16, 17 14, 11, 18 NL 17, 10, 12 12, 13, 18 15, 13, 16 16, 15, 11

- = absence

+ = presence

NL = no bacterial lawn

IL = incomplete bacterial lawn

 

Table 3

Revertant colony counts obtained per plate using S. typhimurium strains TA 98 and TA 100

Strain S. typhimurium	Concentration of test material (µl/plate)	Metabolic activation	Mean revertant colony counts	Individual revertant colony counts
TA 98	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL NL 32 31 29 NL 22 34 34 34	NL NL 30, 33, 34 30, 29, 34 30, 30, 28 NL 19, 21, 27 36, 29, 38 33, 31, 37 31, 33, 37
TA 100	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL NL 69 69 73 NL 44 71 71 68	NL NL 67, 69, 71 62, 69, 75 72, 77, 71 NL 38, 47, 47 75, 69, 69 70, 71, 73 65, 71, 69

- = absence

+ = presence

NL = no bacterial lawn

IL = incomplete bacterial lawn

Table 4

Mutability and sterility tests with S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Strain S. typhimurium	Compound	Concentration of compound (µg/plate)	Metabolic activation	Mean revertant colony counts	Individual revertant colony counts
TA 1535   TA 1537   TA 1538   TA 98   TA 100   TA 1535   TA 1537   TA 1538   TA 98   TA 100   -   -	Sodium azide   4-nitro-o-phenylene-diamine ,,   ,,   Sodium azide   2-amino-anthracene   Neutral red   2-acetyl-aminofluorene   2-amino-anthracene   2-amino-anthracene   S-9 mix  Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one	5   500   ,,   ,,   5   2   10   20   2   2   500 µl   1.0 µl	-   -   -   -   -   +   +   +   +   +       -	825   96   194   2273   745   247   146   709   840   445   0   0	827, 824, 824   80, 92, 117   195, 183, 205   2020, 2328, 2470   716, 771, 749   258, 243, 239   151, 144, 142   697, 705, 725   797, 859, 863   456, 409, 471   0   0

- = absence

+ = presence

Table 5

Validity tests with S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 – colonies per plate

Dilution**	Strain S. typhimurium
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
10^-1D	17	12	17	45	72
10^-6R	*	*	*	*	*
10^-7R	209	520	225	69	521
10^-8R	17	60	38	7	65

 

D = plated onto histidine deficient agar

R = plated onto histidine rich agar

* = too many colonies for accurate counting

** = 3 ml of each dilution per plate

Applicant's summary and conclusion

Conclusions:

No evidence of mutagenic potential of the test chemical was obtained in this bacterial test system at the dose levels used using Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

This study was performed to investigate the potential of the test chemical to induce gene mutation according to Ames metabolic activation test. The test chemical was dissolved in Dimethylsulphoxide (DMSO) and used at dose levels of 10, 1, 0.1, 0.01 µl/well (Bacteriostatic test) and 1, 0.1, 0.01, 0.001 µl/well (Mutation test). The size of zones of inhibition caused by the test chemical with the five tester strains. In the bacteriostatic test, the test chemical showed toxicity towards the bacteria, therefore a top concentration of 1 µl/plate was chosen for the mutation study. At the higher concentrations, the test chemical proved toxic to the cells resulting in either the absence or incomplete formation of a bacterial lawn. No substantial increases in the revertant colony numbers of any of the five strains were observed following treatment with the test chemical at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix) and hence it is not likely to classify as a gene mutant in vitro.