Registration Dossier

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:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Test concentrations with justification for top dose:
5000 µg/plate
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
benzo(a)pyrene
other: 2-Amino-Anthracene

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid

Any other information on results incl. tables

An increase of the number of revertant colonies in the treatment with metabolic activation could be observed towards the bacteria strain TA1535 in three concentrations (5000, 1500 and 500 µg/plate). A concentration-related increase over the tested range was found.

In the treatment without metabolic activation, four concentrations (5000, 1500, 500 and 150  µg/plate) showed an increase of the number of revertant colonies towards the bacteria strain TA1535. In this case, no concentration-related increase over the tested range was found.

 

Therefore, the test item is stated as mutagenic under the test conditions.

To verify the toxicity results, a further experiment was performed with the bacteria strains TA97a, TA98 and TA100 with lower concentrations.

The mean revertant values of the three replicates are presented in the following table.

Table8.1‑a   Mean Revertants Experiment 1a

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

79

90

43

49

78

77

324

339

13

16

sd

13.3

16.4

5.5

9.0

3.5

3.2

45.4

19.7

1.0

4.2

DMSO

Mean

80

99

44

41

86

86

299

347

16

14

sd

24.6

5.7

4.9

9.5

12.7

7.0

37.8

19.7

3.5

3.1

Positive
Controls*

Mean

512

1125

392

227

1256

1312

1200

1163

251

224

sd

72.8

50.8

28.0

40.9

133.1

181.7

104.0

46.9

26.0

90.9

f(I)

6.40

11.36

8.91

5.54

16.10

15.26

4.01

3.35

19.31

16.00

5000 µg/plate

Mean

45

19

0

0

37

26

301

275

165

117

sd

4.0

3.1

0.0

0.0

3.6

4.2

39.3

47.7

53.3

29.5

f(I)

0.56

0.19

0.00

0.00

0.43

0.30

1.01

0.79

10.31

8.36

1500 µg/plate

Mean

16

15

0

0

33

19

261

291

188

101

sd

2.1

1.7

0.0

0.0

1.5

1.2

51.0

56.8

41.8

12.2

f(I)

0.20

0.15

0.00

0.00

0.38

0.22

0.87

0.84

11.75

7.21

500 µg/plate

Mean

51

34

0

0

40

28

317

291

145

58

sd

4.2

4.7

0.0

0.0

6.1

10.0

23.4

34.9

32.3

19.1

f(I)

0.64

0.34

0.00

0.00

0.47

0.33

1.06

0.84

9.06

4.14

150 µg/plate

Mean

75

77

0

0

95

83

241

308

127

20

sd

10.6

15.9

0.0

0.0

20.2

10.5

63.3

52.0

11.5

3.2

f(I)

0.94

0.78

0.00

0.00

1.10

0.97

0.81

0.89

7.94

1.43

50 µg/plate

Mean

81

132

9

9

77

83

225

324

20

16

sd

9.9

6.0

2.1

2.0

11.5

7.6

30.3

45.4

6.1

3.1

f(I)

1.01

1.33

0.20

0.22

0.90

0.97

0.75

0.93

1.25

1.14

f(I) = increase factor, calculation see chapter7.4, page21

Applicant's summary and conclusion

Conclusions:
The test item [3R-(3α, 3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-6-ol (Cedrol) showed an increase in the number of revertants in the bacteria strain TA1535 in experiment 1a.
All negative and nearly all strain-specific positive control values (one marginal exception in experiment 1a) were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that [3R-(3α, 3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-6-ol (Cedrol) is mutagenic in the Salmonella typhimurium test strain TA1535 in the absence and presence of met-abolic activation under the experimental conditions in the present study.
Executive summary:

Two valid experiments were performed.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item[3R-(3α, 3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-6-ol (Cedrol)was tested in theSalmonella typhimuriumreverse mutation assay with five strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the absence and presence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1a:

In this experiment,the test item (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

Signs of toxicity were observed towards the following bacteria strains in the respective concentrations:

·        Bacteria strain TA97a: 5000, 1500 and 500 µg/plate (decrease in the number of revertants)

·        Bacteria strain TA98: 5000, 1500, 500, 150 and 50 µg/plate (decrease in the number of revertants)

·        Bacteria strain TA100: 5000, 1500 and 500 µg/plate (decrease in the number of revertants)

 

The bacterial background lawn was not reduced at any of the concentrations.

The results of this experiment showed that three of the tested concentrations showed an increase in the number of revertants in the tested strain TA1535, in the presence and the absence of metabolic activation.

 


 

Experiment 1b:

Based on the toxicity results of the experiment 1a,the test item was tested up to concentrations of 500 µg/plate in the absence and presence of S9-mix in the bacteria strains TA97a and TA100 and up to concentrations of 50 µg/plate in the absence and presence of S9-mix in the bacteria strain TA98 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

Signs of toxicity were observed in the following concentrations:

·        Bacteria strain TA97a: 500 µg/plate (decrease in the number of revertants)

·        Bacteria strain TA98: 50 µg/plate (decrease in the number of revertants)

Bacteria strain TA100: 500 µg/plate (decrease in the number of revertants)

 

The results of this experiments showed that the test item caused no increase in the number of revertants in these three bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that[3R-(3α, 3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-6-ol (Cedrol)is mutagenic in theSalmonella typhimuriumstrainTA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.