Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Terpineol multiconstituent and alpha-terpineol were found negative in several Ames tests.

Terpineol multiconstituent was also found negative in a recent GLP chromosome aberration test in human lymphocytes conducted according to OECD Guideline 473

Link to relevant study records

Referenceopen allclose all

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.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
Target gene:
Not applicable
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
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.
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'.

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)

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 04 to June 23, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD guideline 473 without any deviation
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultures prepared from the pooled blood of three male donors
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2% S9 fraction of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Range-finder experiment: 5.598-1543 μg/mL (with and without S-9)
Main study:
- Experiment 1: Without S-9: 0, 350, 425 and 450 μg/mL; with S-9: 0, 300, 550 and 625 μg/mL
- Experiment 2: Without S-9: 0, 75, 200 and 225 μg/mL; with S-9: 0, 400, 550, 625 and 650 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation Migrated to IUCLID6: 2.5 and 5 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 10, 20 and 30 µg/mL
Details on test system and experimental conditions:
PREPARATION OF CULTURES: Whole blood cultures pooled from three healthy, non-smoking male volunteers were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 µg/mL gentamycin, so that the final volume following addition of S9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1 °C for approximately 48 hours and rocked continuously.

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 3 or 20 hours, 37 ± 1 ºC
- Fixation time (start of exposure up to harvest of cells): 20 or 20.75 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine, 1 µg/mL for 2 hours
STAIN (for cytogenetic assays): Giemsa (4% v/v)

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: At least 1000 cells/dose were counted in cytotoxicity test to determine the mitotic index; at least 200 metaphase cells/dose were analysed for chromosomal aberrations

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- Cells with structural aberrations including or excluding gaps, polyploidy, hyperdiploidy or endoreduplication were recorded during the study.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range were observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p ≤ 0.05)
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).

- Test article was considered as positive in this assay if all of the above criteria were met.
- Test article was considered as negative in this assay if none of the above criteria were met.
- Results which only partially satisfied the above criteria were dealt with on a case by case basis.
Statistics:
- Statistical method used was Fisher's exact test.
- Proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
- Probability values of p ≤ 0.05 were accepted as significant.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolality: No marked changes in osmolality or pH were observed at the highest concentration tested in the range-finding cytotoxicity experiment (1543 µg/mL, equivalent to 10 mM), compared to the concurrent vehicle controls.
- Solubility/Precipitation: Miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 174.6 mg/mL. Solubility limit in culture medium was approximately 873.1-1746 µg/mL as indicated by precipitation at the higher concentration which persisted for approximately 20 hours after test article addition.

RANGE-FINDING/SCREENING STUDIES:
- In the range-finding cytotoxicity study, precipitation was observed at or above 200 μg/mL and complete cytotoxicity was seen at or above 925.8 µg/mL tested with or without S-9.
- See table 2 for more data

COMPARISON WITH HISTORICAL CONTROL DATA: Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control (normal) ranges.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 2: Range-finder experiment: mitotic index determinations

Treatment

Mitotic index (%)

(µg/mL)

3+17 hours, -S-9

3+17 hours, +S-9

20+0 hours, -S-9

 

A

B

MIH*

A

B

MIH*

A

B

MIH*

Vehicle

5.7

7.4

-

7.0

5.1

-

5.9

6.7

-

5.598

6.2

NT

5

6.7

NT

0

6.0

NT

5

9.330

5.6

NT

15

5.7

NT

6

6.3

NT

0

15.55

7.7

NT

0

5.1

NT

16

5.4

NT

14

25.92

4.5

NT

31

6.2

NT

0

5.3

NT

16

43.19

6.0

NT

8

7.8

NT

0

6.4

NT

0

71.99

6.2

NT

5

6.2

NT

0

6.0

NT

5

120.0

6.8

NT

0

7.1

NT

0

4.9

NT

22

200.0

6.4

NT

2P

4.0

NT

34P

4.5

NT

29P

333.3

6.1

NT

7P

4.8

NT

21P

2.2

NT

65P

555.5

0.0

NT

100P

4.0

NT

34P

0.0

NT

100P

925.8

T

NT

100P

T

NT

100P

T

NT

100P

1543

T

NT

100P

T

NT

100P

T

NT

100P

NT = Not tested; P = Precipitation observed at treatment; T = Toxic

*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

Table 3: Results summary

Treatment

Concentration (mg/mL)

Cytotoxicity (%)

% Cells with Chromosome Aberrations (Excluding Gaps)

Historical (%)#

Statistical significance

Experiment 1

3+17.75 hour -S-9

Vehiclea

-

0.50

0-3

-

 

350.0

0

0.50

 

NC

 

425.0

29

1.00

 

NC

 

450.0

50

0.50

 

NC

 

*NQO, 2.50

ND

8.00

 

p ≤ 0.001

3+17.75 hour +S-9

Vehiclea

-

0.50

0-3

-

 

300.0

0

1.00

 

NC

 

550.0

35

3.00

 

NC

 

625.0

50

5.00

 

NC

 

*CPA, 10.00

ND

25.83

 

p ≤ 0.001

Experiment 2

20+0 hour -S-9

Vehiclea

-

1.00

0-3

-

 

75.00

18

0.00

 

NC

 

200.0

34

1.50

 

NC

 

225.0

53

1.00

 

NC

 

*NQO, 5.00

ND

29.37

 

p ≤ 0.001

3+17 hour +S-9

Vehiclea

-

0.50

0-3

-

 

400.0

7

0.50

 

NC

 

550.0

34

1.50

 

NC

 

625.0

39

2.50

 

NC

 

650.0

50

1.00

 

NC

 

*CPA, 20.00

ND

42.11

 

p ≤ 0.001

a Vehicle control was DMSO

* Positive control

#95th percentile of the observed range

NC = Not calculated

ND = Not determined

Conclusions:
Terpineol multiconstituent is not considered as clastogenic in human lymphocytes according to CLP Regulation (EC) No 1272/2008.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to terpineol multiconstituent in DMSO at concentration range of 5.598-1543 μg/mL, for 3 + 17 h (treatment + recovery) with metabolic activation (2% S-9 fraction of Aroclor 1254-induced male Sprague-Dawley rats), and for 3 + 17 h or 20 + 0 h (treatment + recovery) without metabolic activation for a preliminary cytotoxicity test. In the main test, two experiments were performed at concentrations up to 600 µg/mL without S-9 and up to 800 µg/mL with S-9 and the following concentrations were selected for analysis:

- Experiment 1: Without S-9 (treatment: 3 h): 0, 350, 425 and 450 μg/mL; with S-9 (treatment: 3 h): 0, 300, 550 and 625 μg/mL

- Experiment 2: Without S-9 (treatment: 20 h): 0, 75, 200 and 225 μg/mL; with S-9 (treatment: 3 h): 0, 400, 550, 625 and 650 μg/mL

 

Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control ranges. Positive controls (4-nitroquinoline-N-oxide at 2.5 and 5 µg/mL without S-9 and cyclophosphamide at 10, 20 and 30 µg/mL with S-9) induced the appropriate response. Treatment of cells with terpineol multiconstituent in the presence or absence of S-9 in both experiments resulted in frequencies of cells with structural or numerical aberrations that were generally similar to those observed in concurrent vehicle controls for all concentrations analysed. Numbers of aberrant cells (excluding gaps) in treated cultures fell within the normal range with the exception of one culture at the highest concentration analysed with S-9 in experiment 1 (625.0 µg/mL). However, the aberration frequency (excluding gaps) in the replicate culture at 625.0 µg/mL in experiment 1 and in all other cultures analysed in experiments 1 and 2 fell within the normal range.

 

Therefore, terpineol multiconstituent is not considered as clastogenic in human lymphocytes according to CLP Regulation (EC) No 1272 /2008.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Alpha-terpineol is one of the main constituents of multiconstituent substance TERPINEOL MULTICONSTITUENT. Therefore, data on alpha-terpineol can be used for extrapolation to TERPINEOL MULTICONSTITUENT.
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
an alteration of the background lawn at 2000 µg/plate with S9 and a decrease in revertant number at 1250 µg/plate without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
other: TA 97a, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
an alteration of the background lawn at 2000 and 2500 µg/plate for TA 100 and TA 98 with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
no data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table: 2 Toxicity of alpha terpineol compound to S.typhimurium TA 100 strain

Dose (µg /plate)

-S9

+S9

3000

-

0 / 3

2750

-

-

2500

-

180 ± 11

2000

68/0/0

184 ± 8

1500

126 ± 35

-

1250

145 ± 6

-

1000

164 ± 5

-

900

-

-

800

-

-

700

-

-

600

-

-

500

-

-

400

-

-

300

-

-

200

-

-

0

167 ± 6

166 ± 2

PC

913 ± 65

464 ± 35

Table 3: Mutagenicity testing of alpha terpineol in the Salmonella / microsome assay [TA100, TA98, TA97a and TA102 tester strains]

Dose (µg /plate)

TA 100

TA 98

TA 97a

TA 102

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

2500

-

141 ± 62*

-

4 /0/0

-

-

-

-

2000

53 ± 14*

146 ± 2

-

68 ± 13

-

272 ± 34

-

833 ± 57*

1500

165 ± 16

212 ± 28

43 ± 5

60 ± 10

225
± 32

248 ± 12

-

1573 ± 191

1250

158 ± 19

188 ± 23

42 ± 2

58 ± 2

224 ± 11

246 ± 6

862 ± 187

1727 ± 108

1000

176 ± 6

208 ± 33

40 ± 8

54 ± 10

191 ± 8

254 ± 21

1004 ± 488

1429 ± 46

750

177 ± 14

196 ± 1

36 ± 4

69 ± 10

216 ± 16

246 ± 12

1418 ±  176

1177 ± 42

500

177 ± 12

161 ± 24

28 ± 7

53 ± 10

195 ± 13

241 ± 16

1312 ± 89

1116 ± 74

250

186 ± 9

-

30 ± 6

-

182 ± 12

-

812 ± 166

790 ± 47

100

-

-

-

-

-

-

924 ± 151

-

50

-

-

-

-

-

-

737 ± 32

-

25

-

-

-

-

-

-

611 ± 65

-

0

176 ± 14

197 ± 7

40 ± 7

70 ± 6

158 ± 2

214 ± 18

731 ± 36

771 ± 37

PC

898 ± 51

742 ± 13

192 ± 31

312 ± 33

1098 ± 80

870 ± 140

4875 ± 1031

2024 ± 182

Dose O- Negative control: 100 µl ethanol PA; PC-Positive control

(-) Dose not tested.

(*) Toxicity apparent as an alternation of the background lawn

(/+) mutant counts of individual plates

Conclusions:
alpha-Terpineol was found mutagenic with and without S9 in TA 102 strain but was non mutagenic with and without S9 in TA97a, TA98 and TA100 strains.
Executive summary:

In a reverse gene mutation assay in bacteria performed in non GLP conditions but similarly to OECD guideline 471, TA97a, TA98, TA100 and TA102 strains of S. typhimurium were exposed to alpha-terpineol at concentrations between 0 and 2500 µg/plate in the presence and absence of mammalian metabolic activation system (lyophilized rat liver S9 fraction induced by Aroclor 1254). 

alpha-Terpineol was tested for cytotoxicity at different dose concentrations. The toxicity appeared at 2500 µg/plate with S9 and at 2000 µg/plate without S9 for TA100 strain. The positive controls induced the appropriate responses in the corresponding strains.

alpha-Terpineol caused dose-related increase in the number of histidine revertants with TA102 tester strain with and without S9. There was no evidence of concentration related positive response for induced mutant colonies over background in the other strains with and without S9.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Alpha-terpineol is one of the main constituents of multiconstituent substance TERPINEOL MULTICONSTITUENT. Therefore, data on alpha-terpineol can be used for extrapolation to TERPINEOL MULTICONSTITUENT.
Reason / purpose:
read-across source
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:
not specified
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
alpha-Terpineol is not considered as mutagenic in the bacterial system according to CLP Regulation (EC) No 1272/2008.
Executive summary:

In a reverse gene mutation assay in bacteria performed similarly to OECD Guideline 471, strains of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 100 and TA 98) were exposed to alpha-terpineol up to 10000 µg or nL/plate in both the absence and presence of metabolic activation (S9 fraction of Aroclor 1254-induced adult male Sprague-Dawley rat liver) according to the direct plate incorporation method.

 

Alpha terpineol showed no substantial increases in revertant colony numbers obtained with any of the tester strains up to the highest concentration tested in either presence or absence of S9 mix.

 

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

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Alpha-terpineol is one of the main constituents of multiconstituent substance TERPINEOL MULTICONSTITUENT. Therefore, data on alpha-terpineol can be used for extrapolation to TERPINEOL MULTICONSTITUENT.
Reason / purpose:
read-across source
Key result
Species / strain:
other: TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
no data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
alpha-Terpineol was not mutagenic in the Ames test in both plate incorporation and preincubation methods with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria conducted similarly to OECD guideline 471, TA98, TA100, TA1535, TA1537 and TA1538 strains of S. typhimurium  were exposed to alpha-terpineol at concentrations between 1µg and 1000 µg/plate in the presence and absence of mammalian metabolic activation system liver S9 homogenate, from male Sprague-Dawley rats and Syriyan golden hamsters injected with Aroclor 1254 at 500 mg/kg body weight.

alpha-Terpineol was tested for mutagenicity at different dose concentrations with both direct plate incorporation and preincubation methodology. alpha-Terpineol caused no dose-related response in the number of histidine auxotroph revertants. The positive controls induced the appropriate responses in the corresponding strains.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Alpha-terpineol is one of the main constituents of multiconstituent substance TERPINEOL MULTICONSTITUENT. Therefore, data on alpha-terpineol can be used for extrapolation to TERPINEOL MULTICONSTITUENT.
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The doses of chemical selected for testing were with in the range yielding approximately 0-90 % cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
no data
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Test results confirmed previous results showing negative response both with and without metabolic activation.
Executive summary:

In a mammalian cell gene mutation assay conducted similarly to OECD guideline 476, mouse lymphoma L5178Y cells cultured in vitro were exposed to alpha-terpineol at concentrations between 0.14 µg/mL and 0.65 µg/mL in the presence and absence of metabolic activation with liver S9 prepared from Aroclor 1254-induced male Sprangue-Dawley rats. 

alpha-Terpineol was tested for cytotoxic concentration up to an upper limit of 10000 µg/plate. In both nonactivated and S9-activated conditions, response was negative at a dose 0.14-0.65 µg/mL The positive controls ethylmethylsulfonate (without metabolic activation) and 3-methylcholanthrene (with metabolic activation) induced the appropriate response.

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Terpineol multiconstituent and alpha-terpineol were found negative in several Ames tests with and without metabolic activation. A GLP study on terpineol multiconstituent conducted according to OECD guideline 471 was identified as key study.

Terpineol multiconstituent was also found negative in a recent GLP chromosome aberration test in human lymphocytes conducted according to OECD guideline 473, identified as key study.

alpha-Terpineol was negative in a gene mutation test conducted similarly to OECD guideline 476 using mouse lymphoma tk+/- L5178Y cells with and without metabolic activation.


Justification for classification or non-classification

Terpineol multiconstituent was negative in an Ames test and in a chromosome aberration test. alpha-Terpineol showed similar mutagenicity properties as terpineol multiconstituent in several Ames tests and was found negative in an in vitro gene mutation test in mammalian cells. Therefore, terpineol multiconstituent is considered as non mutagenic and is not classified as mutagenic according to CLP Regulation (EC) No 1272/2008.