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Diss Factsheets

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:
weight of evidence
Study period:
20 September 2012 – 22 March 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Terpineol
EC Number:
232-268-1
EC Name:
Terpineol
Cas Number:
8000-41-7
Molecular formula:
C10H18O
IUPAC Name:
2-(4-methylcyclohex-3-en-1-yl)propan-2-ol

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium and tryptophan-requiring gene in Escherichia coli
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from male Sprague-Dawley rat liver and induced by Phenobarbital (PB) and 5,6-benzoflavone (BF)
Test concentrations with justification for top dose:
Preliminary experiment: 19.5, 78.1, 313, 1250 and 5000 μg/plate.
In the preliminary test, growth inhibition was observed at 1250 μg/plate for all strains without metabolic activity. When tested with metabolic activity, growth inhibition was observed at 313 μg/plate for strains TA 98, TA 1535 and TA 1537, and at 1250 μg/plate for strains TA 100 and E. Coli.
The highest dose selected for the main experiments for each strain was the lowest dose showing growth inhibition in the preliminary test. This dose was further diluted 5 times with the ratio of 2 to obtain 6 doses.

Main tests 1 and 2 (all strains -S9 and TA 98, TA 1535 and TA 1537 +S9): 39.1, 78.1, 156, 313, 625 and 1250 μg/plate
Main tests 1 and 2 (TA 100 and E. Coli +S9): 9.77, 19.5, 39.1, 78.1, 156 and 313μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to solubility in water of 1.98 g/L, a solubility test was performed on DMSO. As a result, the test substance was dissolved in DMSO at 50 mg/mL and no reactivity such as generation of heat or gas was observed, so the test was conducted using DMSO as a solvent.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Methoxy-6-chloro-9-[ 3-(2-chloro ethyl)-aminopropylamino] acridine 2HCL (ICR-191); 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation: 0.1 mL of solvent or positive control or test solution, 0.5 mL of S9 mix or 0.1 M phosphate buffer solution (pH 7.4) and 0.1 mL of culture solution of each strain were added to the test tube. Preincubation was carried out with shaking (80 rpm) for 20 minutes at 37ºC. After completion of the preincubation 2.0 mL of top agar were added and after stirring the mixture was uniformly layered on a minimum glucose agar plate medium. Once confirmed that the top agar layered on the minimum glucose agar plate medium was solidified, the plate was turned upside down, placed in the incubator, and incubate at 37ºC for 49 hours in preliminary test and for 48.5 h in the main tests.

- Cell density at seeding (if applicable): 1 x 10^9 cells/mL.

DURATION
- Preincubation period: 20 min.
- Exposure duration: 48.5 h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants (revertant bacteria) can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition. The presence or absence of growth inhibition was observed using a stereoscopic microscope.
Evaluation criteria:
A result was considered positive when the number of revertant colonies in the test substance treated group showed an increase of 2 times or more as compared with the number of spontaneous revertant colonies (negative control value) and a dose response and reproducibility were observed. Also, a positive result was decided when a clear dose response was not shown but an increase of more than twice the number of spontaneously reversed mutant colonies was counted and when reproducibility was confirmed in two main tests.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 625 μg/plate and above)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 625 μg/plate and above)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 313 μg/plate and above)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 1250 μg/plate and above)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 625 μg/plate and above)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 313 μg/plate and above)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 625 μg/plate and above)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 313 μg/plate and above)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Results of Test 1 (-S9Mix)

Metabolic activation

Dose of test substance

(μg/plate)

Number of revertants (colony count / plate)

Base pair substitution type

Frameshift type

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

S9 Mix (-)

Negative control (DMSO)

100

115

120 (112±10.4)

12

13

9 (11±2.1)

29

23

25 (26±3.1)

19

13

13 (15±3.5)

8

5

7 (7±1.5)

39.1

90

99

97 (95±4.7

5

7

5 (6±1.2)

26

31

38 (32±6.0)

15

11

13 (13±2.0)

4

5

6 (5±1.0)

78.1

97

95

108 (100±7.0)

7

12

10 (10±2.5)

23

27

25 (25±2.0)

13

12

13 (13±0.6)

6

5

4 (5±1.0)

156

100

103

99 (101±2.1)

5

9

5 (6 ±2.3)

36

29

42 (36±6.5)

10

12

15 (12±2.5)

8

5

11 (8±3.0)

313

98

101

99 (99±1.5)

5

6

11 (7±3.2)

22

30

36 (29±7.0)

13

10

11 (11±1.5)

6

4

7 (6±1.5)

625

46*

50*

47* (48±2.1)

0*

0*

0* (0±0)

27

21

27 (25±3.5)

8*

8*

8* (8±0.0)

0*

0*

0* (0±0)

1250

0*

0*

0* (0±0)

0*

0*

0* (0±0)

0*

0*

0* (0±0)

0*

0*

0* (0±0)

0*

0*

0* (0±0)

Positive controls

S9Mix (without)

Name

AF-2

SAZ

AF-2

AF-2

ICR-191

Dose

(μg/plate)

0.01

0.5

0.01

0.1

1.0

Number of colonies/ plate

712

690

641 (681± 36.3)

358

304

374 (345±36.7)

115

123

117 (118±4.2)

461

428

425 (438±20.0)

1993

1905

1810 (1903±91.5)

Notes:

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

SAZ: Sodium azide

ICR-191: 2-Methoxy-6-chloro-9-[ 3-(2-chloro ethyl)-aminopropylamino] acridine · 2HCL

*: Growth inhibition by the test substance was observed.

( ): The inside shows the average value and standard deviation of three plates

Table 2: Results of Test 1 (+S9Mix)

Metabolic activation

Dose of test substance

(μg/plate)

Number of revertants (colony count / plate)

Base pair substitution type

Frameshift type

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

S9 Mix (+)

Negative control (DMSO)

114

123

138 (125±12.1)

8

9

7 (8±1.0)

30

30

33 (31±1.7)

22

27

26 (25±2.6)

6

7

10 (8±2.1)

9.77

NT

11

10

7 (9±2.1)

NT

20

20

19 (20±0.6)

8

7

10 (8±1.5)

19.5

NT

9

8

9 (9±0.6)

NT

18

21

26 (22±4.0)

8

6

9 (8±1.5)

39.1

116

121

110 (116±5.5)

5

11

10 (9±3.2)

22

31

38 (30±8.0)

24

23

20 (22±2.1)

5

8

8 (7±1.7)

78.1

116

100

109 (108±8.0)

11

10

8 (10±1.5)

33

24

29 (29±4.5)

21

20

24 (22±2.1)

9

11

7 (9±2.0)

156

110

91

111 (104±11.3)

8

11

8 (9±1.7)

26

25

23 (25±1.5)

17

18

23 (19±3.2)

7

6

8 (7±1.0)

313

123

112

105 (113±9.1)

5*

7*

7* (6±1.2)

31

27

39 (32±6.1)

22*

24*

18* (21±3.1)

5*

7*

8* (7±1.5)

625

90*

91*

103* (95±7.2)

NT

21

34

23 (26±7.0)

NT

NT

1250

0*

0*

0* (0±0.0)

NT

0*

0*

0* (0±0.0)

NT

NT

Positive controls

S9Mix (with)

Name

B[α] P

2AA

2AA

B[α] P

B[α] P

Dose

(μg/plate)

5.0

2.0

10.0

5.0

5.0

Number of colonies/ plate

782

731

725 (746 ± 31. 3)

359

288

344 (330±37.4)

556

717

633 (635±80.8)

338

337

376 (350±22.2)

58

55

77 (63±11.9)

Notes:

B[α] P: Benzo [α] pyrene

2AA: 2-Aminoanthracene

*: Growth inhibition by the test substance was observed.

NT: not tested

( ): The inside shows the average value and standard deviation of three plates.

Table 3: Results of Test 2 (-S9Mix)

Metabolic activation

Dose of test substance

(μg/plate)

Number of revertants (colony count / plate)

Base pair substitution type

Frameshift type

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

S9 Mix (-)

Negative control (DMSO)

103

104

116 (108±7.2)

10

7

10 (9±1.7)

38

38

41 (39±1.7)

24

21

19 (21±2.5)

7

10

8 (8±1.5)

39.1

85

91

112 (96±14.2)

10

13

12 (12±1.5)

39

36

33 (36±3.0)

22

20

20 (21±1.2)

6

7

10 (8±2.1)

78.1

92

88

95 (92±3.5)

10

10

12 (11±1.2)

39

49

38 (42±6.1)

21

22

16 (20±3.2)

8

8

8 (8±0.0)

156

116

97

111 (108±9.8)

7

10

7 (8±1.7)

44

44

28 (39±9.2)

18

21

20 (20±1.5)

15

10

11 (12±2.6)

313

104

117

112 (111±6.6)

6

10

8 (8±2.0)

41

38

47 (42±4.6)

19

13

18 (17±3.2)

10

10

9 (10±0.6)

625

53*

44*

69* (55±12.7)

2*

5*

3* (3±1.5)

34

31

30 (32±2.1)

10*

11*

10* (10±0.6)

0*

0*

0* (0±0)

1250

0*

0*

0* (0±0)

0*

0*

0* (0±0)

0*

0*

0* (0±0)

0*

0*

0* (0±0)

0*

0*

0* (0±0)

Positive controls

S9Mix (without)

Name

AF-2

SAZ

AF-2

AF-2

ICR-191

Dose

(μg/plate)

0.01

0.5

0.01

0.1

1.0

Number of colonies/ plate

637

692

650 (660± 28.7)

293

309

353 (318±31.1)

118

102

106 (109±8.3)

439

428

474 (447±24.0)

1548

1594

1489 (1544±52.6)

Notes:

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

SAZ: Sodium azide

ICR-191: 2-Methoxy-6-chloro-9-[ 3-(2-chloro ethyl)-aminopropylamino] acridine · 2HC1

*: Growth inhibition by the test substance was observed.

( ): The inside shows the average value and standard deviation of three plates.

Table 4: Results of Test 2 (+S9Mix)

Metabolic activation

Dose of test substance

(μg/plate)

Number of revertants (colony count / plate)

Base pair substitution type

Frameshift type

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

S9 Mix (+)

Negative control (DMSO)

120

108

114 (114±6.0)

14

10

11 (12±2.1)

42

44

30 (39±7.6)

27

27

28 (27±0.6)

13

10

10 (11±1.7)

9.77

NT

10

7

10 (9±1.7)

NT

28

25

22 (25±3.0)

10

7

13 (10±3.0)

19.5

NT

7

10

6 (8±2.1)

NT

29

27

24 (27±2.5)

9

7

8 (8±1.0)

39.1

105

119

100 (108±9.8)

11

11

10 (11±0.6)

42

47

43 (44±2.6)

25

30

27 (27±2.5)

10

10

13 (11±1.7)

78.1

125

114

116 (118±5.9)

8

10

13 (10±2.5)

51

40

39 (43±6.7)

31

23

29 (28±4.2)

7

6

7 (7±0.6)

156

106

122

123 (117±9.5)

8

9

6 (8±1.5)

54

42

53 (50±6.7)

30

29

29 (29±0.6)

7

11

10 (9±2.1)

313

119

133

114 (122±9.8)

8*

10*

12* (10±2.0)

39

37

41 (39±2.0)

26*

27*

23* (25±2.1)

8*

7*

7 (7±0.6)

625

90*

104*

75* (90±14.5)

NT

38

38

47 (41±5.2)

NT

NT

1250

0*

0*

0* (0±0.0)

NT

0*

0*

0* (0±0.0)

NT

NT

Positive controls

S9Mix (with)

Name

B[α] P

2AA

2AA

B[α] P

B[α] P

Dose

(μg/plate)

5.0

2.0

10.0

5.0

5.0

Number of colonies/ plate

727

771

804 (767 ± 38.6)

379

339

352 (357±20.4)

852

841

890 (861±25.7)

288

266

253 (269±17.7)

79

82

93 (85±7.4)

Notes:

B[α] P: Benzo [α] pyrene

2AA: 2-Aminoanthracene

*: Growth inhibition by the test substance was observed.

NT: not tested

( ): The inside shows the average value and standard deviation of three plates.

Applicant's summary and conclusion

Conclusions:
Terpineol did not show any mutagenic effect in bacteria under test conditions with and without metabolic activation.

Executive summary:

Terpineol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, 1535 and TA1537 and Escherichia coli strain WP2 uvrA with and without metabolic activation (S9). The experiment was performed according to OECD guideline 471 with GLP. Based on growth inhibition examined in a preliminary test up to a maximum dose of 5000μg/plate, tested concentrations in the main test were: 39.1, 78.1, 156, 313, 625 and 1250μg/plate for all strains without metabolic activationand for strains TA 98, TA 1535 and TA 1537 with metabolic activation, and 9.77, 19.5,39.1, 78.1, 156 and 313μg/plate for strains TA 100 and E. Coli WP2 with metabolic activation. Two main tests were performed in triplicate using preincubation method and DMSO as solvent. Negative and positive controls were within background data of the test laboratory. Under test conditions, terpineol was found not mutagenic in all strains tested with and without metabolic activation.