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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
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

Reference
Name:
Unnamed
Type:
Constituent
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: Solid (yellow)

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
Controlsopen allclose all
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
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
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.