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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November - 08 December 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD Guideline 471 without deviations.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pentan-2-ol; 6-(2,2,3-trimethylcyclopent-3-en-1-yl)hexan-3-ol
EC Number:
939-525-3
Cas Number:
1471313-03-7
Molecular formula:
C14H26O
IUPAC Name:
3-methyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pentan-2-ol; 6-(2,2,3-trimethylcyclopent-3-en-1-yl)hexan-3-ol
Details on test material:
- Name of test material (as cited in study report): Sandalore
- Physical state: Colourless liquid
- Analytical purity: sum of C14H16O isomers 92.6 %
- Lot/batch No.: 9000399614
- Expiration date of the lot/batch: 30 September 2002
- Storage condition of test material: Approximately 4 °C

Method

Target gene:
His+ for S. typhimurium
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: see table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
15 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of male Wistar Hanlbm rats induced with phenobarbital (80 mg/kg bw, intraperitoneal route) and β-Naphthoflavone (oral route)
Test concentrations with justification for top dose:
Pre-experiment for toxicity: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate, with and without S9 mix in TA 98 and TA 100 strains (plate incorporation and pre-incubation methods)

Main experiment (with and without S9 mix):
Experiment 1 (plate incorporation method) and Experiment 2 (pre-incubation method):
- 33, 100, 333, 1000, 2500 and 5000 µg/plate in TA 98 and TA 100 strains
- 3, 10, 33, 100, 333 and 1000 µg/plate in TA 1535 and TA 1537 strains
- 10, 33, 100, 333, 1000 and 2500 µg/plate in TA 102 strain
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Purity: > 99 %
- Source: MERCK, Darmstadt, Germany
- Justification for choice of solvent/vehicle: DMSO was chosen as solvent because of its solubility properties and its relative non-toxicity to the bacteria.
- Formulation procedure: On the day of experiment, the test item was dissolved in the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (10 µg/plate in TA 100 and TA 1535); 4-nitro-o-phenylene-diamine (10 µg/plate in TA 98 and 50 µg/plate in TA 1537); methyl methane sulfonate (5 µL/plate in TA 102)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2.5 µg/plate in TA 100, TA 1535, TA 1537 and TA 98; 10 µg/plate in TA 102)
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM:
- TA 1535, TA 1537, TA 100 and TA 102 strains: Ames' Laboratory (University of California, Berkeley, USA)
- TA 98 strain: von E. Merck (Darmstadt, Germany)

METHOD OF APPLICATION: In agar (plate incorporation and preincubation method)

DURATION
- Preincubation period: 60 minutes at 37 °C
- Incubation period: 48 h at 37 °C for both plate incorporation and preincubation methods

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of spontaneous revertants or a clearing of the bacterial background lawn.

OTHER:
- Colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, Karben, Germany)
- Pre-experiment was reported as part of the main experiment 1.
Evaluation criteria:
- Test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
- Test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at anyone of the test points is considered non-mutagenic in this system.

Biologically relevant response is described as follows:
- Test item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and TA 102 or thrice in strains TA 1535 and TA 1537.
- Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the criteria described above or not.
Statistics:
None

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- Toxic effects were evident as the reduction of number of revertant colonies with the test item compared to vehicle control.
- Plates with the test item showed normal background growth up to 5000 µg/plate in strain TA 98 and TA 100.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data (generated since 1995)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects, evident as a reduction in the number of revertants were observed at the following concentrations:
Experiment 1:
- Without S9 mix: 333 and 1000 µg/plate (TA 1535 and TA 1537), 5000 µg/plate (TA 98), 100-5000 µg/plate (TA 100), 2500 µg/plate (TA 102)
- With S9 mix: 1000 µg/plate (TA 1537), 5000 µg/plate (TA 98), 333-5000 µg/plate (TA 100), 2500 µg/plate (TA 102)

Experiment 2:
- Without S9 mix: 333 and 1000 µg/plate (TA 1535), 100-1000 µg/plate (TA 1537), 5000 µg/plate (TA 98), 100-5000 µg/plate (TA 100)
- With S9 mix: 333 and 1000 µg/plate (TA 1537), 333-5000 µg/plate (TA 100)

- In experiment 1, a reduction in the number of revertants was also observed without S9 mix in strain TA 1535 at 33 µg/plate and in strain TA 1537 at 10 µg/plate. This reduction is judged to be based upon statistical fluctuations at such low numbers of bacteria and does not represent a true toxic effect.
- Plates incubated with the test item showed normal background growth up to the highest concentration with and without S9 mix in all strains used.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Summary of results – without S9 mix

Concentration (µg/plate)

Mean number of revertants per plate in Experiment 1 and 2

TA 1535

TA 1537

TA 98

TA 100

TA 102

1

2

1

2

1

2

1

2

1

2

Negative control

10

10

6

8

19

21

104

135

134

174

Solvent control

12

9

6

7

22

24

103

114

131

170

Positive control#

581

611

49

49

180

226

703

746

706

363

3

6

9

4

6

-

-

-

-

-

-

10

5

7

1

5

-

-

-

-

88

170

33

5

7

4

4

12

20

66

66

110

152

100

6

8

4

2

11

19

47

40

113

145

333

4

4

2

1

8

19

39

37

80

145

1000

3

0

1

0

14

16

17

35

74

130

2500

-

-

-

-

17

17

11

26

66

105

5000

-

-

-

-

10

11

7

24

-

-

# Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100

4-nitro-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate)

Methyl methane sulfonate (5 µg/plate) strain TA 102

Table 7.6.1/3: Summary of results – with S9 mix

Concentration (µg/plate)

Mean number of revertants per plate in Experiment 1 and 2

TA 1535

TA 1537

TA 98

TA 100

TA 102

1

2

1

2

1

2

1

2

1

2

Negative control

10

9

6

6

40

40

121

112

217

297

Solvent control

10

13

6

6

44

34

131

120

195

258

Positive control*

473

99

83

100

627

432

800

560

759

764

3

10

9

5

5

-

-

-

-

-

-

10

7

8

6

4

-

-

-

-

146

263

33

10

13

5

5

39

34

121

114

189

282

100

9

7

5

4

38

32

95

93

170

255

333

7

8

3

2

30

28

48

57

186

226

1000

6

8

0

0

35

32

28

34

172

191

2500

-

-

-

-

26

23

22

11

107

157

5000

-

-

-

-

19

23

17

12

-

-

*2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98 and TA 100

2-aminoanthracene (10.0 µg/plate) strain TA 102

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, the substance is not considered as mutagenic in S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102).
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to the substance at the following concentrations:

Pre-experiment for toxicity: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate, with and without S9 mix in TA 98 and TA 100 strains (plate incorporation and pre-incubation methods)

Main experiment (with and without S9 mix): Experiment 1 (plate incorporation method) and Experiment 2 (pre-incubation method):

- 33, 100, 333, 1000, 2500 and 5000 µg/plate in TA 98 and TA 100 strains

- 3, 10, 33, 100, 333 and 1000 µg/plate in TA 1535 and TA 1537 strains

- 10, 33, 100, 333, 1000 and 2500 µg/plate in TA 102 strain

Metabolic activation system used in this test was 15 % (v/v) S9 mix. S9 fraction were prepared from liver homogenates of male Wistar Hanlbm rats induced with phenobarbital (80 mg/kg bw, intraperitoneal route) and β-Naphthoflavone (oral route). Negative (untreated), vehicle and positive control groups were also included in this test.

Test substance did not induce precipitation. Toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains used. The negative, vehicle and positive controls induced the appropriate responses in the corresponding strains. The test item showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S9 mix.

Under the test conditions, the substance is not considered as mutagenic in this bacterial system.