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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
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted: 21' July 1997, OECD/OCDE Publication Service, Paris
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-5-isopropyl-2-methylcyclohexa-1,3-diene
EC Number:
224-167-6
EC Name:
(R)-5-isopropyl-2-methylcyclohexa-1,3-diene
Cas Number:
4221-98-1
Molecular formula:
C10H16
IUPAC Name:
(5R)-2-methyl-5-(propan-2-yl)cyclohexa-1,3-diene

Method

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:
S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
5- 5000 µg per plate presence of S9-mix
1.5- 5000 µg per plate absence of S9-mix
Vehicle / solvent:
- Vehicle used: Ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours at 37°C

NUMBER OF REPLICATIONS: The experiment was repeated in full after an interval of at least 3 days.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
A reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity.
According Ames et al. (1975) as well as reported by De Serres and Shelby (1979).
Statistics:
XE2-test (Mohn and Ellenberger, 1977)

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9-mix at 50 µg/plate, with 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 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 mix at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
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:
no cytotoxicity, but tested up to precipitating concentrations
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the absence of a metabolizing system (Experiment 1)

 

S9 mix

TA98

TA100

TA102

TA1535

TA1537

Control

0 µg/plate

-

13

113

273

12

14

Solvent control

0 µg/plate

-

17

101

293

11

12

Test substance in µg/plate

 

5

-

15

-

277

14

10

15

-

19

108

296

13

11

50

-

19

93

268

9

9

150

-

11

96

131 (T)

6

8

500

-

16

85

99 (T)

8

6

1500

-

13

86

117 (T)

9

8

5000 (P)

-

12

89

-

-

-

NaN3 (0.7 µg/plate)

-

-

314

-

292

-

2-NF (2.5 µg/plate)

-

395

-

-

-

-

9-AA (50 µg/plate)

-

-

-

-

-

381

Mitomycin C (0.15 µg/plate)

-

-

-

1003

-

-

P: precipitation of test compound

T: bacteriotoxic

Solvent control: Ethanol, 50 µL/plate

2-NF: 2-nitrofluorene

9-AA: 9-aminoacridine

Table 2: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the presence of a metabolizing system (Experiment 1)

 

S9 mix

TA98

TA100

TA102

TA1535

TA1537

Control

0 µg/plate

+

22

96

294

7

15

Solvent control

0 µg/plate

+

22

101

290

15

15

Test substance in µg/plate

 

15

+

19

101

278

17

13

50

+

21

97

188

17

12

150

+

23

101

222

15

15

500

+

17

80

209

17

14

1500

+

22

89

226

17

10

5000 (P)

+

16

93

NA

15

13

2-AA (0.8 µg/plate)

+

501

839

683

123

 

2-AA (1.7µg/plate)

 

-

-

 

 

314

Solvent control: Ethanol, 50 µL/plate

2-AA: 2-aminoanthracene

Table 3: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the absence of a metabolizing system (Experiment 2)

 

S9 mix

TA98

TA100

TA1535

TA1537

Control

0 µg/plate

-

23

84

24

8

Solvent control

0 µg/plate

-

23

76

23

10

Test substance in µg/plate

 

5

-

21

-

-

-

15

-

14

86

20

10

50

-

23

80

27

8

150

-

19

79

21

6

500

-

12

76

20

10

1500

-

18

79

21

5

5000 (P)

-

-

86

22

6 (T)

NaN3 (0.7 µg/plate)

-

-

283

817

-

2-NF (2.5 µg/plate)

-

258

 

-

-

9-AA (50 µg/plate)

-

 

 

-

844

T: bacteriotoxic

Table 4: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the presence of a metabolizing system (Experiment 2)

 

S9 mix

TA98

TA100

TA102

TA1535

TA1537

Control

0 µg/plate

+

28

90

284

12

13

Solvent control

0 µg/plate

+

27

85

285

18

14

Test substance in µg/plate

 

5

+

-

-

324

-

-

15

+

25

-

337

-

14

50

+

29

85

325

17

13

150

+

15

82

226

17

13

500

+

26

84

247

18

7

1500

+

27

79

-

16

9

5000 (P)

+

28

83

-

14

14

2-AA (0.8 µg/plate)

+

583

511

-

136

-

2-AA (0.9 µg/plate)

 

 

 

747

 

-

2-AA (1.7µg/plate)

 

 

 

-

 

233

Table 5: Induction of revertants (mean of 3 replicates) in S. typhimurium strain TA 102 in the absence of a metabolizing system (Experiment 2)

 

S9 mix

TA102

Control

0 µg/plate

-

309

Solvent control

0 µg/plate

-

301

Test substance in µg/plate

-

-

1.5

-

286

5

-

298

15

-

281

50

-

140 (T)

150

-

142 (T)

500

-

135 (T)

Mitomycin C (0.15 µg/plate)

-

959

T: bacteriotoxic

Applicant's summary and conclusion

Conclusions:
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.
 
Executive summary:

In the GLP and guideline compliant study the test substance was tested in reverse mutation study with the stains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100, TA 102). 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 ethanol and tested in concentrations of 5 to 5000 µg/plate in the presence and of 1.5 to 5000 µg per plate absence of S9.

The test substance was bacteriotoxic in the absence of S9-mix towards the strains TA 102 at 50 µg/plate and towards the strain TA 1537 at 5000 µg/plate.

In the presence of S9-mix the test substance was bacteriotoxic towards the strains TA 102 at 500 µg/plate. Precipitation of the test compound on the plates was observed at 5000 µg/plate.

 

The positive and negative controls were all valid and in the expected ranges.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.

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.