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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:
key study
Study period:
Feb 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(Commission Directive 2000/32/EC)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol
EC Number:
947-716-8
Molecular formula:
C15H30O
IUPAC Name:
Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/ß-naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test:
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation in TA100

Experiment 1 and 2:
0, 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation in all strains
Vehicle / solvent:
- Vehicle/solvent used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene (2AA); 1,8-Dihydroxyanthraquinone (DAN)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for experiment 1 and 2

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Rationale for test conditions:
The test conditions were chosen according to OECD 471.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results was considerd first, statistical methods, as recommended by the UKEMS (Kirkland D.J., Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press, 1989) were also used as an aid to evaluation, however, statistical significance was not the only determining factor for a positive response. A test material was considered non-mutagenic (negative) in the test system if the above criteria were not met.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
greasy precipitate ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
greasy precipitate ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
greasy precipitate ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
greasy precipitate ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
greasy precipitate ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A greasy precipitate was observed at and above 1500 μg/plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The dose range of 50 to 5000 µg/plate for both main experiments was determined in a preliminary toxicity assay in S. typhimurium strain TA 100.

HISTORICAL CONTROL DATA: Positive and vehicle controls were within the normal range of historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.

Any other information on results incl. tables

Spontaneous mutation rates for the negative controls were considered to be acceptable. The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial stains, with any dose of the test substance, either with or without metabolic activation.

Table 2. Results of Experiment 1

EXPERIMENT 1

S9-Mix

Without

 

Test substance (µg/plate)

TA 100

TA 1535

TA 102

TA 98

TA 1537

SC

114 ± 7.4

23 ± 4.0

332 ± 14.1

21 ± 5.6

16 ± 3.5

50

109 ± 19.5

22 ± 0.6

293 ± 39.7

24 ± 6.7

13 ± 3.0

150

99 ± 3.8

21 ± 4.6

256 ± 41.6

22 ± 6.8

15 ± 3.6

500

96 ± 17.7

21 ± 4.7

265 ± 9.3

19 ± 5.5

12 ± 2.3

1500

105 ± 22.7 P

20 ± 4.4 P

242 ± 34.1 P

20 ± 2.1 P

13 ± 4.6 P

5000

85 ± 11.0 P

23 ± 1.2 P

264 ± 33.0 P

21 ± 1.2 P

12 ± 2.3 P

ENNG

479 ± 50.8

382 ± 25.7

 

 

 

MMC

 

 

1739 ± 6.5

 

 

4NQO

 

 

 

275 ± 16.8

 

9AA

 

 

 

 

756 ± 230.1

S9-Mix

With

 

Test substance (µg/plate)

TA 100

TA 1535

TA 102

TA 98

TA 1537

SC

88 ± 6.4

14 ± 0.0

328 ± 14.2

32 ± 5.0

19 ± 3.5

50

93 ± 6.5

14 ± 1.7

336 ± 30.1

32 ± 1.7

18 ± 4.7

150

89 ± 18.6

10 ± 2.3

285 ± 23.1

30 ± 5.5

20 ± 2.1

500

 88 ± 12.5

13 ± 4.2

308 ±19.0

28 ± 5.5

14 ± 6.7

1500

83 ± 7.6 P

10 ± 2.3 P

296 ± 27.1 P

23 ± 5.9 P

11 ± 0.6 P

5000

89 ± 9.6 P

13 ± 2.5 P

286 ± 11.0 P

26 ± 2.3 P

12 ± 3.5 P

2AA

2762 ± 93.6

174 ± 42.2

 

 

210 ± 19.6

DAN

 

 

1010 ± 101.7

 

 

BP

 

 

 

158 ± 36.7

 

SC = Solvent Control (DMSO)

P = Precipitate

Positive Controls: ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine; MMC: Mitomycin C; 4NQO: 4-Nitroquinoline-1-oxide; 9-AA: 9-aminoacridine; 2AA: 2-Aminoanthracene; DAN: 1,8-Dihydroxyanthraquinone; BP: Benzo(a)pyrene

Table 3. Results of Experiment 2

EXPERIMENT 2

S9-Mix

Without

 

Test

substance

(µg/plate)

TA 100

TA 1535

TA 102

TA 98

TA 1537

SC

92 ± 13.9

25 ± 5.3

293 ± 33.0

21 ± 1.0

13 ± 5.9

50

98 ± 17.1

19 ± 5.2

321 ± 17.6

14 ± 5.7

13 ± 3.8

150

103 ± 5.3

19 ± 2.6

284 ± 8.5

11 ± 3.5

11 ± 2.6

500

91 ± 13.1

9 ± 3.5

272 ± 16.2

12 ± 4.0

8 ± 1.5

1500

78 ± 7.4 P

12 ± 4.2 P

288 ± 22.5 P

9 ± 0.6 P

11 ± 1.5 P

5000

83 ± 14.2 P

14 ± 5.1 P

274 ± 7.5 P

8 ± 2.0 P

17 ± 2.3 P

ENNG

673 ± 76.4

859 ± 123.0

 

 

 

MMC

 

 

1341 ± 95.8

 

 

4NQO

 

 

 

128 ± 28.4

 

9AA

 

 

 

 

323 ± 32.5

S9-Mix

With

 

Test

substance

(µg/plate)

TA 100

TA 1535

TA 102

TA 98

TA 1537

SC

84 ± 12.7

11 ± 2.1

310 ± 30.7

15 ± 4.2

11 ± 3.5

50

84 ± 9.8

17 ± 2.0

315 ± 10.1

17 ± 1.2

10 ± 3.1

150

81 ± 4.0

9 ± 4.0

283 ± 16.5

20 ± 6.1

7 ± 2.1

500

61 ± 7.8

11 ± 0.0

291 ± 20.4

17 ± 5.1

8 ± 1.0

1500

57 ± 5.5 P

10 ± 4.2 P

268 ± 12.1 P

14 ± 2.6 P

11 ± 2.9 P

5000

59 ± 7.0 P

10 ± 2.3 P

272 ± 12.6 P

14 ± 3.6 P

10 ± 3.6 P

2AA

1536 ± 162.9

174 ± 52.5

 

 

568 ± 46.2

DAN

 

 

733 ± 22.6

 

 

BP

 

 

 

173 ± 17.7

 

SC = Solvent Control (DMSO)

P = Precipitate

Positive Controls: ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine; MMC: Mitomycin C; 4NQO: 4-Nitroquinoline-1-oxide; 9-AA: 9-aminoacridine; 2AA: 2-Aminoanthracene; DAN: 1,8-Dihydroxyanthraquinone; BP: Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames assay the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation.
Executive summary:

A bacterial gene mutation assay (Ames) with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2016). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 50 to 5000 µg/plate were selected for the incubation with and without metabolic activation in both experiments. Precipitation occurred at and above 1500 µg/plate. The test substance was not bacteriotoxic at any dose and strain. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tested strains in the presence and absence of metabolic activation. The vehicle and positive control data were in the range of the historical control data of the laboratory. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains in the presence and absence of metabolic activation.

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