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Toxicological information

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:
7 March 2006 - 9 May 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study in compliance with OECD guideline 471 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: 049807
Purity: 67.2% (sum of the three main constituents)
Name of test material (as cited in study report): TERPINEOL MULTICONSTITUENT
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 30 November 2017

Method

Target gene:
Not applicable
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: bacterial broth medium
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: bacterial broth medium
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fraction from Sprague Dawley rats treated with beta-naphtoflavone and phenobarbital
Test concentrations with justification for top dose:
Cytotoxicity test: 50, 150, 500, 1500 and 5000 µg/plate
Main test: 19, 56, 167, 500 and 1500 µg/plate
Vehicle / solvent:
DMSO
- Justification for choice of solvent/vehicle: test substance not soluble in aqueous vehicles
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period (before treatment): 13 h at 37°C
- Preincubation period (with test substance) in the second main experiment with S9: 30 min
- Exposure duration: 48 h at 37 °C
- Expression time (cells in growth medium): 48 h at 37 °C

SELECTION AGENT (mutation assays): minimum medium

NUMBER OF REPLICATIONS:
- cytotoxicity test: 1
- main test: 3

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants

OTHER: 2 mL agar, 0.1 mL of test substance dilution, 0.1 mL of bacterial culture and 0.5 mL of S9 mix (when appropriate) were plated on solid minimum medium
Evaluation criteria:
- statistically significant ratio (revertants with test substance / revertants with solvent) higher than 2 for TA 98, TA 100 and TA 102, and higher than 3 for TA 1535 and TA 1537
- increase in the number of revertants linked with the increase in test substance concentration
- reproducible results
Statistics:
Dunett's test on revertants with test substance / revertants with solvent ratios.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate for all strains and slightly to strong at 1500 µg/plate depending on the strains
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
strong at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test substance was observed

RANGE-FINDING/SCREENING STUDIES: see table 4 for cytotoxicity results
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: results of the first main experiment

Strain

Compound

Dose level (µg/plate)

S9 mix

Mean revertant colony counts

SD

Ratio treated/solvent

Individual revertant colony counts

TA 98

DMSO

 

 

 

 

 

2 nitrofluorene

 

DMSO

 

 

 

 

 

2-aminoanthracene

 

19

56

167

500

1500

1

 

 

19

56

167

500

1500

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

19

18

17

17

17

5

319

 

22

23

25

25

24

20

2653

4

2

3

3

4

5

46

 

5

6

7

4

7

4

150

 

0.9

0.9

0.9

0.9

0.3 *

 

 

 

1.0

1.1

1.1

1.1

0.9

 

23, 16, 19

19, 20, 16

14, 18, 20

19, 19, 14

13, 21, 17

3, 2, 11

320, 364, 273

 

27, 22, 18

16, 27, 25

29, 17, 29

30, 22, 23

18, 23, 32

24, 19, 17

2520, 2624, 2816

TA 100

DMSO

 

 

 

 

 

Sodium azide

 

DMSO

 

 

 

 

 

2-aminoant-racene

 

19

56

167

500

1500

1.5

 

 

19

56

167

500

1500

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

74

81

79

90

85

60

155

 

94

137

140

105

120

102

3397

6

6

16

12

12

17

15

 

12

3

3

3

14

9

411

 

1.1

1.0

1.2

1.1

0.8

 

 

 

1.5 *

1.5 *

1.1

1.3 *

1.1

 

71, 81, 70

74, 86, 82

65, 76, 97

79, 102, 89

95, 71, 88

79, 46, 55

141, 154, 171

 

102, 80, 101

135, 140, 136

140, 137, 142

106, 107, 102

105, 125, 131

107, 108, 92

3152, 3872, 3168

TA 102

DMSO

 

 

 

 

 

Mitomycine C

 

DMSO

 

 

 

 

 

Benzo(a)pyrene

 

19

56

167

500

1500

0.125

 

 

19

56

167

500

1500

4

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

192

193

225

125

73

0

1339

 

214

232

266

158

153

5

436

8

30

8

50

29

0

368

 

16

35

23

9

32

3

26

 

1.0

1.2

0.7 *

0.4 *

0.0 *

 

 

 

1.1

1.2

0.7 *

0.7 *

0.0 *

 

188, 186, 201

228, 172, 180

232, 216, 228

156, 68, 152

63, 51, 106

1060, 1200, 1756

 

 

208, 232, 201

212, 272, 212

288, 268, 242

158, 167, 149

176, 167, 116

3, 4, 8

464, 414, 429

TA 1535

DMSO

 

 

 

 

 

Azide sodium

 

DMSO

 

 

 

 

 

2-aminoanthracene

 

19

56

167

500

1500

1.5

 

 

19

56

167

500

1500

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

13

13

14

16

10

2

685

 

11

6

8

9

9

4

111

4

1

1

7

4

2

24

 

2

1

2

1

1

1

35

 

1.1

1.1

1.3

0.8

0.1 *

 

 

0.5 *

0.7 *

0.8

0.8

0.3 *

14, 8, 16

13, 13, 14

14,14, 15

9, 22, 17

5, 12, 12

0, 4, 1

658, 704, 694

 

11, 12, 9

6, 6, 5

7, 10, 6

8, 9, 9

8, 8, 10

3, 4, 4

149, 81, 103

TA 1537

DMSO

 

 

 

 

 

9-aminoacridine

 

DMSO

 

 

 

 

 

2-aminoanthracene

 

19

56

167

500

1500

40

 

 

19

56

167

500

1500

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

10

6

7

10

6

7

957

 

7

6

4

9

8

3

192

6

4

2

1

2

2

93

 

3

1

1

2

3

3

28

 

0.5

0.7

1.0

0.5

0.6

 

 

 

0.8

0.6

1.3

1.1

0.4 *

 

17, 5, 9

3, 4, 10

5, 7, 9

10, 10, 11

7, 4, 6

9, 6, 5

1002, 1018, 850

 

10, 5, 7

5, 6, 7

4, 4, 5

7, 11, 10

6, 7, 12

6, 2, 1

161, 202, 214

* statistically significant (p<0.05)

Table 3: results of the second main experiment

Strain

Compound

Dose level (µg/plate)

S9 mix

Mean revertant colony counts

SD

Ratio treated/solvent

Individual revertant colony counts

TA 98

DMSO

 

 

 

 

 

nitro-2-fluorene

 

DMSO

 

 

 

 

 

2-aminoanthracene

 

19

56

167

500

1500

1.5

 

 

19

56

167

500

1500

1

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

22

22

15

22

16

6

360

 

21

28

28

27

13

2

1229

5

4

2

2

3

2

60

 

9

6

2

9

6

4

85

 

1.0

0.7 *

1.0

0.7

0.3 *

 

 

 

1.3

1.3

1.3

0.6

0.1 *

 

23, 16, 26

24, 24, 17

16, 15, 13

23, 20, 24

19, 13, 15

8, 5, 6

316, 428, 336

 

30, 13, 20

33, 22, 28

29, 29, 26

35, 27, 18

20, 8, 11

0, 0, 7

1140, 1308, 1240

TA 100

DMSO

 

 

 

 

 

Sodium azide

 

DMSO

 

 

 

 

 

2-aminoanthracene

 

19

56

167

500

1500

1.5

 

 

19

56

167

500

1500

1

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

90

80

67

90

78

54

201

 

94

92

97

129

82

36

808

8

8

7

12

18

1

20

 

3

8

19

23

16

49

78

 

0.9

0.7 *

1.0

0.9

0.6 *

 

 

 

1.0

1.0

1.4

0.9

0.4 *

 

92, 82, 97

72, 87, 80

72, 59, 70

91, 78, 101

59, 80, 95

54, 54, 55

223, 185, 195

 

92, 93, 97

98, 83, 95

114, 100, 77

102, 138, 146

64, 95, 86

0, 16, 92

812, 884, 728

TA 102

DMSO

 

 

 

 

 

Mitomycine C

 

DMSO

 

 

 

 

 

Benzo(a)pyrene

 

19

56

167

500

1500

0.125

 

 

19

56

167

500

1500

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

180

205

159

154

105

4

1131

 

107

137

110

98

93

0

214

47

33

15

10

8

4

110

 

10

4

19

14

19

0

18

 

1.1

0.9

0.9

0.6 *

0.0 *

 

 

 

1.3 *

1.0

0.9

0.9

0.0 *

 

228, 178, 134

228, 168, 220

175, 147, 154

150, 147, 165

95, 110, 109

0, 8, 4

1192, 1004, 1196

 

100, 118, 103

142, 136, 134

93, 131, 106

113, 85, 95

76, 88, 114

0, 0, 0

218, 229, 194

TA 1535

DMSO

 

 

 

 

 

Azide sodium

 

DMSO

 

 

 

 

 

2-aminoanthracene

 

19

56

167

500

1500

1.5

 

 

19

56

167

500

1500

1

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

13

16

18

13

11

6

647

 

9

11

10

5

3

0

218

2

2

2

1

3

2

50

 

3

2

3

2

2

0

12

 

1.2

1.3

1.0

0.8

0.4 *

 

 

1.2

1.1

0.6

0.3 *

0.0 *

15, 12, 13

15, 15, 18

19, 18, 16

12, 13, 13

11, 8, 14

7, 7, 3

670, 590, 682

 

6, 10, 12

10, 11, 13

13, 9, 8

7, 5, 4

2, 5, 1

0, 0, 0

205, 229, 219

TA 1537

DMSO

 

 

 

 

 

9-aminoacridine

 

DMSO

 

 

 

 

 

2-aminoanthracene

 

19

56

167

500

1500

40

 

 

19

56

167

500

1500

1

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

10

7

10

6

7

3

336

 

6

9

7

7

9

0

45

1

4

3

4

4

3

118

 

2

0

1

2

14

0

6

0.7

1.0

0.6

0.6

0.3 *

 

 

 

1.4

1.2

1.1

1.5

0.0 *

 11, 11, 9

12, 4, 5

13, 10, 8

11, 4, 4

3, 10, 7

0, 3, 6

468, 242, 298

 

5, 8, 6

9, 9, 9

7, 8, 7

5, 7, 8

26, 2, 0

0, 0, 0

38, 49, 48

* statistically significant (p<0.05)

Applicant's summary and conclusion

Conclusions:
Terpineol multiconstituent was found not to be mutagenic in TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, strains of S. typhimurium (TA 98, TA 100, TA 102, TA 1535 and TA 1537) were exposed to Terpineol multiconstituent at concentrations of 50, 150, 500, 1500 and 5000 µg/plate in both the absence and presence of S9 metabolic activation according to the direct plate incorporation method for a preliminary cytotoxicity test. In the main test, two experiments were performed at 19, 56, 167, 500 and 1500 µg/plate (up to cytotoxic concentrations) in both the absence and presence of S9. These experiments were performed according to the direct plate incorporation method except for the second experiment with S9 which was performed with a pre incubation period of 30 min with the test substance and S9 mix.

The positive controls induced the appropriate responses in the corresponding strains. Terpineol multiconstituent showed no biologically significant 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.

This study is classified as acceptable and satisfies the requirement for bacterial reverse gene mutation endpoint.

Therefore, Terpineol multiconstituent is not considered as mutagenic in this bacterial system according to CLP Regulation (EC) No 1272/2008.