Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2000-11-08 to 2000-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol
EC Number:
947-711-0
Molecular formula:
C14H28O
IUPAC Name:
Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: besides being histidine auxotrophic, all strains carry an additional mutation (loss of outer lipopolysaccharide barrier) which increases cell permeability
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: distilled water and DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Remarks:
9-Aminoacridine, mitomycin C, and sodium azide were dissolved in distilled water. 2-Aminoanthracene, and 2-nitrofluorene were dissolved in DMSO.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 parallel plates (2 independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting of the number of revertant colonies (his+ revertants)

OTHER EXAMINATIONS:
- Examination for the existente of a normal background lawn and/or precipitates and microscopically for microcolony growth
Evaluation criteria:
The number of spontaneous revertants observed using each of the five strains and the results with the positive controls were compared to historical control data. Differences between the number of revertants in the negative controls and the test plates were tested for significance.
Statistics:
Estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level was performed using a X2-test.

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:
cytotoxicity
Remarks:
with S9 mix - at 5000 µg/plate; without S9 mix - at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with and without S9 mix - at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 mix - at 5000 µg/plate; without S9 mix - at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 mix - at 1500 µg/plate; without S9 mix - at 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 mix - at 500 µg/plate; without S9 mix - at 150 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation of the test substance on the plates was observed at 5000 μg/plate. The test substance failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. The X2 test did not reveal a significant effect at any of the test points.

HISTORICAL CONTROL DATA
- historical overview of the revertant frequencies of the strains used of the years 1998 to 2000
- TA1535 -S9: 30 ± 14, +S9: 19 ± 5
- TA1537 -S9: 10 ± 4, +S9: 16 ± 5
- TA98 -S9: 29 ± 8, +S9: 40 ± 10
- TA100 -S9: 134 ± 31, +S9: 118 ± 26
- TA102 -S9: 279 ± 41, +S9: 307 ± 45

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of three plates), Experiment 1

Substance

conc. [µg/plate]

strain

TA 98

Strain

TA 100

strain

TA 102

strain

TA 1535

strain

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Control

0

18

24

133

129

309

284

22

10

10

11

Solvent control

0

17

24

123

122

250

268

23

10

9

13

Test item 1

5

20

25

 

 

251

292

 

 

 

 

Test item 2

15

15

21

102

 

259

295

 

 

 

 

Test item 3

50

17

23

92

126

205

299

28

11

6

14

Test item 4

150

17

28

101

119

193

270

16

10

7

14

Test item 5

500

16

21

100

95

171 T

204 T

10

9

6

11

Test item 6

1500

17

22

87 T

98

95 T

161 T

6 T

7

4 T

9

Test item 7

5000 P

8 T

15 T

 

92 T

 

 

5 T

6 T

4 T

4 T

Sodium azide
NaN3

0.7

 

 

560

 

 

 

817

 

 

 

2-Nitrofluorene

2-NF

2.5

283

 

 

 

 

 

 

 

 

 

9-Aminoacridine
9-AA

50

 

 

 

 

 

 

 

 

583

 

Mitomycin C

0.15

 

 

 

 

959

 

 

 

 

 

2-aminoanthracene
2-AA

0.8
0.9

1.7

 

687

 

 

 

1035

 

 

 

 

747

 

 

207

 

 

 

 

 

231

-MA: absence of metabolic activation

+MA: presence of metabolic activation

P: precipitation of test item

T: bacteriotoxic

solvent control: DMSO, 50 µL/plate

Table 2: Number of revertants per plate (mean of three plates), Experiment 2

Substance

conc. [µg/plate]

strain

TA 98

Strain

TA 100

strain

TA 102

strain

TA 1535

strain

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Control

0

24

24

115

123

351

293

16

9

14

15

Solvent control

0

21

22

106

106

326

308

20

10

16

15

Test item 1

5

 

 

112

 

273

294

19

 

10

 

Test item 2

15

 

 

92

99

270

287

22

 

13

15

Test item 3

50

16

18

98

124

205

270

20

13

9

14

Test item 4

150

14

20

81

120

168 T

246

14

9

9

16

Test item 5

500

9

19

71 T

113

150 T

224 T

17

9

8

13

Test item 6

1500

10 T

19

 

83 T

 

 

 

6

 

7 T

Test item 7

5000 P

12 T

14 T

 

 

 

 

 

7

 

 

Sodium azide
NaN3

0.7

 

 

317

 

 

 

789

 

 

 

2-Nitrofluorene

2-NF

2.5

355

 

 

 

 

 

 

 

 

 

9-Aminoacridine
9-AA

50

 

 

 

 

 

 

 

 

333

 

Mitomycin C

0.15

 

 

 

 

855

 

 

 

 

 

2-aminoanthracene
2-AA

0.8
0.9

1.7

 

453

 

 

 

735

 

 

 

383

 

 

 

141

 

 

 

 

 

197

-MA: absence of metabolic activation

+MA: presence of metabolic activation

P: precipitation of test item

T: bacteriotoxic

solvent control: DMSO, 50 µL/plate

Applicant's summary and conclusion

Conclusions:
The test substance was not mutagenic in the bacterial reverse mutation assay.
Executive summary:

The genetic toxicity of the test item was tested according to OECD 471 and Regulation (EC) No. B13/14. The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98; TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of S9 mix. The test substance was basteriotoxic towards all tested strain at different concentration levels in the presence and absence of S9 mix. Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the tested concentration range, the test substance did not induce a significant increase in the mutation frequency of the tester strains with and without metabolic activation. In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.