Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in all strains tested (OECD TG 471)

Cytogenicity in mammalian cells: negative in cultured human lymphocytes (OECD TG 473)

Mutagenicity in mammalian cells: negative in L5178Y cells (OECD TG 476)

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA (TSCA) OPPTS harmonised guidelines
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine (Salmonella); tryptophan (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
5-5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test material was insoluble in water but fully soluble in DMSO at 50 mg/ml
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation for: E. coli (2 µg/plate), TA 100 (3 µg/plate), TA 1535 (5 µg/plate)
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation for: TA 1537 (80 µg/plate)
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation for TA 98 (0.2 µg/plate)
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation for: TA 100 (1 µg/plate), TA 1535 and TA 1537 (2 µg/plate), E. coli (10 µg/plate)
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation for TA 98 (5 µg/plate)
Details on test system and experimental conditions:
ACTIVATION: Phenobarbitol/beta-naphthoflavone induced rat liver S9: 10% in S9 mix including NADP and glucose-6-phosphate. 0.5 ml of S9 mix added to 2 ml agar, 0.1 ml bacterial culture and 0.1 ml test substance before pouring plates.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF REPLICATIONS: triplicate plates; experiment repeated: Experiment 1 plate incorporation; Experiment 2 preincubation

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in background lawn; reduction in number of revertants per plate
Evaluation criteria:
A substance is considered positive if it induces a dose-related increase in revertant frequency over the dose range and/or a reproducible increase at one or more concentration in at least one bacterial strain with or without metabolic activation.
Statistics:
Statistical evaluation used as aid to evaluation if necessary.
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:
500 µg/plate (preincubation, with metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no information
- Effects of osmolality: no information
- Evaporation from medium: no information
- Water solubility: not soluble in water
- Precipitation: none recorded
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Slight thinning of background lawn was observed in range-finding study

COMPARISON WITH HISTORICAL CONTROL DATA: results were within the historical control values

ADDITIONAL INFORMATION ON CYTOTOXICITY: Experiment 1: slight thinning of bacterial background lawn was observed in all tester strains with and without metabolic activation, initially at 500 µg/plate. Experiment 2: reduction in bacterial background lawn at 15 µg/plate in absence of metabolic activation, and at 500 µg/plate with metabolic activation.
Remarks on result:
other: No mutagenic potential

Table 1 Experiment 1 Plate incorporation: Number of revertants per plate (mean of 3 plates)

Concentration µg/plate

TA 100

TA 1535

E. coli SP2uvrA

TA 98

TA 1537

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

0***

110

93

19

14

16

19

18

26

5

9

5

111

96

21

14

NT

NT

21

26

5

4

15

108

87

19

9

18

20

13

21

9

8

50

100

100

20

14

20

27

25

25

6

9

150

121

91

12

8

19

23

20

26

4

7

500

127

106

25

12

21

25

17

23

11*

8*

1500

124*

113*

27*

22*

23

18

15*

28

7**

9**

5000

133*

92*

32*

20*

23

15

15*

20*

7**

9**

Positive control

730

1250

606

254

655

333

152

178

737

215

* Sparse bacterial lawn

** Very sparse bacterial lawn

*** Solvent control with DMSO

NT not tested

 

Table 2a Experiment 2 Pre-incubation: Number of revertants per plate (mean of 3 plates)

Concentration µg/plate

TA 100

TA 1535

TA 98

TA 1537

Concentration µg/plate

TA 100

TA 1535

E. coli WP2uvrA

TA 98

TA 1537

- MA

- MA

- MA

- MA

+ MA

+ MA

+ MA

+ MA

+ MA

0***

112

19

19

13

0***

98

11

43

24

9

0.05

109

17

20

11

1.5

87

10

NT

26

10

0.15

103

21

18

9

5

88

13

NT

27

11

0.5

99

22

19

11

15

79

10

42

25

9

1.5

107

18

19

11

50

91

9

38

21

11

5

104

21

18

13

150

84

10

31

22

8

15

96*

22*

15*

9*

500

0**

10*

34

20

10*

50

93*

22*

11*

4*

1500

0**

0**

33

0

1**

-

-

-

-

-

5000

NT

NT

33*

NT

NT

Positive control

489

1719

139

769

Positive control

1017

287

341

360

314

* Sparse bacterial lawn

** Very sparse bacterial lawn

*** Solvent control with DMSO

NT not tested

 

 

Table 2b Experiment 2 Pre-incubation: Number of revertants per plate (mean of 3 plates)

Concentration µl/plate

E. coli WP2uvrA

- MA

0***

337

15

31

50

30

150

29

500

34

1500

31*

5000

24*

Positive control

1027

* Sparse bacterial lawn

*** Solvent control with DMSO

Conclusions:
Turpentine oil has been tested according to OECD 471 and under GLP. No increase in the number of revertants per plate was observed in any of the bacterial strains used when tested with and without metabolic activation using the plate incorporation method. The results were confirmed in the repeat assay using the preincubation method. Positive and solvent controls gave the expected results. It is concluded that turpentine oil is negative for mutagenicity to bacteria under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 April 2010 to 20 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitol/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1: 1.25 - 20 µg/ml (-S9), 5 - 70µg/ml (+S9). Experiment 2 5 - 60 µg/ml (-S9); 5 - 50 µg/ml (+S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility of test material. Maximum dose limited by toxicity of solvent to 2500 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation (400 µg/ml experiment 1, 150 µg/ml experiment 2)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation (2 µg/ml)
Details on test system and experimental conditions:
ACTIVATION S9 mix included NADP and glucose-6-phosphate as cofactors. Final concentration of S9 in cultures: 2%
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (without activation experiment 1, with activation experiments 1 and 2); 24 hours (without activation experiment 2)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days


SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: Duplicate exposure cultures, experiment repeated.

NUMBER OF CELLS EVALUATED: 2000 cells per culture evaluated for mutant frequency

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth and relative total growth

OTHER EXAMINATIONS:
- Other: large and small colony distribution
Evaluation criteria:
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
Statistical significance was determined using the UKEMS statistical package.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
50 μg/ml (with metabolic activation, 4 and 24 h exposure)
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: no marked increase in pH
- Effects of osmolality: osmolality did not increase by more than 50 mOsm
- Evaporation from medium: no information
- Water solubility: not soluble in water
- Precipitation: none observed
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Indicated toxicity to test material at 19.53 to 39.06 μg/ml

COMPARISON WITH HISTORICAL CONTROL DATA: vehicle and positive controls were within the range of historical values

ADDITIONAL INFORMATION ON CYTOTOXICITY: In experiment 1, less cytotoxicity was observed than expected in the absence of metabolic activation. However, based on the levels of toxicity observed in the Preliminary Toxicity Test and the very steep toxicity curve of the test material, it is considered that a slightly higher dose level may have resulted in an excessive level of toxicity. Therefore, with no evidence of any toxicologically significant increases in mutant frequency in the 24-hour exposure group in the absence of metabolic in Experiment 2, where very near optimum levels of toxicity were achieved or exceeded at higher dose concentrations, it is considered that the test material had been adequately tested.
Remarks on result:
other: No mutagenic potential

Experiment 1: A slightly elevated mutant frequency was noted at a dose that exceeded the acceptable toxicity limits. However, the GEF was not exceeded, the mutant frequency value observed was within the acceptable range for vehicle controls, there was no evidence of any marked increase in absolute numbers of mutant colonies, and there was no shift towards small colony formation that would have indicated a clastogenic response. It was considered that the increase in mutant frequency observed at this dose level was due to a cytotoxic mechanism and not a true genotoxic response and, therefore, was of no toxicological significance. It should also be noted that a decrease in viability was observed at this dose level, indicating that modest residual toxicity had occurred. The excessive levels of toxicity observed at and above 60 μg/ml in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance.

Experiment 2: The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in the presence of metabolic activation. The test material induced a very modest statistically significant dose related (linear-trend) increase in the mutant frequency x 10-6 per viable cell in the absence of metabolic activation (Table 9). However, statistically significant increases in mutant frequency were not observed at any of the individual dose levels, the GEF was not exceeded at any of the individual dose levels with acceptable levels of toxicity or dose levels where near optimum levels of toxicity where achieved or marginally exceeded, there was no evidence of any marked increases in absolute numbers of mutant colonies, and the mutant frequency values observed were in the acceptable range for vehicle controls. Modest increases in mutant frequency were observed at 40 and 45 μg/ml, (dose levels that had been excluded from the statistical analysis due to excessive toxicity). However, as was seen in the 4-hour exposure group in the presence of metabolic activation in the first experiment, it was considered that the increases in mutant frequency observed at these dose levels was due to a cytotoxic mechanism and not a true genotoxic response and were of no toxicological significance. Therefore, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels in either the first or second experiment, including dose levels where optimum / near optimum levels of toxicity were achieved or exceeded, it is considered that the test material had been adequately tested.

Table 1 Preliminary Toxicity Test. The results for the Relative Suspension Growth (%RSG)

Dose

(μg/ml)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

9.77

88

101

74

19.53

9

99

75

39.06

0

55

2

78.13

0

0

0

156.25

0

0

0

312.5

0

0

0

625

0

0

0

1250

0

0

0

2500

0

0

0

 

Table 2 Results of mutagenicity study, experiment 1 (mean of 2 treatments)

Treatment

(μg/ml)

4-Hours-S-9

Treatment

(μg/ml)

4-Hours+S-9

%RSG

RTG

MF§

%RSG

RTG

MF§

0

100

1

75.74

0

100

1

80.87

1.25

96

not plated

5

88

1.02

89.77

2.5

103

not plated

10

89

1

73.13

5

100

1.06

91.48

20

82

0.79

100.65

10

93

1.11

87.04

30

68

0.73

113.87

12.5

87

1.03

66.96

40

58

0.49

75.59

15

80

0.98

72.15

50 *

6

0.05

172.74

17.5

57

0.7

72.85

60

0

not plated

20

37

0.36

61.73

70

0

not plated

Linear trend

Not significant

Linear trend

Not significant

EMS

400

71

0.46

705.10

CP

2

54

0.28

846.58

 

Table 3 Results of mutagenicity study, experiment 2 (mean of 2 treatments)

Treatment

(μg/ml)

4-Hours-S-9

Treatment

(μg/ml)

4-Hours+S-9

%RSG

RTG

MF§

%RSG

RTG

MF§

0

100

1

108.03

0

100

1

120.23

5

98

1.02

146.10

5

97

1.43

77.14

10

97

1.24

104.79

10

100

1.64

76.37

20

81

0.81

163.05

20

94

1.40

74.88

30

48

0.58

125.99

30

71

0.98

109.58

35

28

0.29

160.54

35

42

0.57

107.95

40*

11

0.06

206.67

40

20

0.31

90.12

45*

1

0.01

242.78

45

3

not plated

50

0

not plated

50

1

not plated

55

0

not plated

-

-

-

-

60

0

not plated

-

-

-

-

Linear trend

Not significant

Linear trend

Not significant

EMS

400

68

0.40

1524.73

CP

2

70

0.58

634.89

* Treatment excluded from statistics due to toxicity

EMS Ethylmethanesulphonate

CP Cyclophosphamide
Conclusions:
Turpentine oil has been tested according to OECD 476 and under GLP. No toxicologically significant increases in the mutant frequency at the TK +/- locus in L 5178Y cells were observed in the presence or absence of metabolic activation after 4 h exposure in the initial experiment. The result was confirmed in the repeat experiment when the cells were exposed to the test organism for 24 h in the absence of metabolic activation, and 4 h in the presence of metabolic activation. It is concluded that turpentine oil is negative for mutagenicity in L5178Y cells under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I: 0.032 - 5.0 µl/ml (with and without S9); Experiment II: 0.006 to 0.93 µl/ml (without S9), 0.30 to 5.0 µl/ml (with S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours (about 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures, experiment repeated

NUMBER OF CELLS EVALUATED: At least 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.17 µl/ml , 22 h exposure
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item Turpentine oil CAS 8006-64-2, dissolved in THF, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 5.0 µl/ml was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In this study, no precipitation of the test item in the culture medium was observed. A decrease in the osmolarity and no relevant increase in the pH value was observed (Exp. I: solvent control: 496 mOsm, pH 7.6 versus 316 mOsm and pH 7.6 at 5.0 µl/ml; Exp. II: solvent control: 387 mOsm, pH 7.5 versus 360 mOsm and pH 7.5 at 0.93 µg/ml). Phase separation was observed in Experiment I at 1.63 µl/ml and above in the absence and presence of S9 mix and in Experiment II at 0.93 µl/ml in the absence and at 0.93 µLl/ml and above in the presence of S9 mix.
In Experiment I in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II in the absence of S9 mix, cytotoxicity was observed at the two highest evaluated concentrations (54.3, 49.1 % of control). In Experiment I in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. However, in Experiment II in the presence of S9 mix the highest applied concentration was not evaluable due to low metaphase numbers.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix the aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were in the range of the solvent control values (0.5 – 3.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data. In Experiment II after continuous treatment with the test item the lowest evaluated concentration 0.032 µg/ml slightly exceeded the laboratory’s historical control range. Since no statistical significance and no dose-dependency were observed the finding is not biologically relevant.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (825.0 µg/ml) or CPA (7.5 or 15.0 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
Remarks on result:
other: No mutagenic potential

Summary of results of the chromosomal aberration study with Turpentine oil CAS 8006-64-2

Preparation

interval

 

Test item

concentration in µl/ml

Mitotic indices

in % of control

Aberrant cells

in %

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs without S9 mix. Experiment 1

22 hrs

 

 

 

 

Solvent control1

100.0

1.0

1.0

0.0

Positive control2

51.6

11.0

10.5S

3.5

0.099

95.5

1.5

1.0

0.0

0.17

94.1

1.5

1.5

1.0

0.30

70.3

1.5

1.5

0.0

Exposure period 22 hrs without S9 mix. Experiment 2

22 hrs

 

 

 

 

 

Solvent control1

100.0

2.0

1.5

0.0

Positive control2

40.3

27.5

25.0S

2.5

0.032#

84.8

3.8

3.0

0.0

0.057

86.8

4.0

2.0

0.0

0.099

54.3

2.0

1.5

0.0

0.17

49.1

1.0

0.5

0.0

Exposure period 4 hrs with S9 mix Experiments 1 and 2

22 hrs, expt 1

 

 

 

 

Solvent control1

100.0

0.5

0.5

0.0

Positive control3

63.9

12.5

12.0S

3.0

1.63

99.7

0.5

0.0

0.0

2.86

102.6

0.5

0.5

0.0

5.00

81.0

1.0

1.0

0.0

22 hrs, expt 2

 

 

 

 

Solvent control1

100.0

3.5

3.0

0.5

Positive control4

51.0

8.5

8.5S

0.5

0.93

86.6

1.5

1.5

0.0

1.63

91.9

0.5

0.5

0.0

2.86

91.0

1.5

0.5

0.0

*  Including cells carrying exchanges

#   Evaluation of 200 metaphases per culture

S  Aberration frequency statistically significant higher than corresponding control values

1   THF 0.5 % (v/v)

2     EMS825.0 µg/ml

3   CPA  15.0 µg/ml

4   CPA    7.5 µg/ml

Conclusions:
Turpentine oil CAS 8006-64-2 has been tested in a valid study according to OECD TG 473 under GLP. Under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro in the initial experiment, nor in the repeat experiment with longer exposure. The vehicle and positive controls gave expected results. Therefore, Turpentine oil CAS 8006-64-2 is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest evaluable concentrations.
Executive summary:

The test item Turpentine oil CAS 8006-64-2, dissolved in THF, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I & II

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were scored for structural chromosomal aberrations.

The highest applied concentration in the pre-test on toxicity (5.0 µL/mL of the test item) was chosen with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473.

In Experiment I in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II in the absence of S9 mix, cytotoxicity was observed at the two highest evaluated concentrations. In Experiment I in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the presence of S9 mix the highest applied concentration was not evaluable due to low metaphase numbers.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro

Information is available for TOPP from reliable studies for all the required in vitro endpoints. The results of all the studies were in agreement.

TOPP (CAS 8006-64-2) has been tested in a study conducted according to OECD test guideline 471 and under GLP (Bowles, 2010). No evidence of mutagenic activity was observed. Mutagenicity was tested using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and Escherichia coli WP2uvrA. The tests were conducted with and without metabolic activation (phenobarbitone/β-naphthoflavone-induced rat liver S9 mix).

TOPP (CAS 8006-64-2) has been tested in a study conducted according to OECD test guideline 476 and under GLP (Flanders, 2010). No evidence of mutagenic activity was observed. Mutagenicity was tested using mouse lymphoma L5178Y cells. The tests were conducted with and without metabolic activation (phenobarbital and β-naphthoflavone-induced rat liver S9 mix).

TOPP (CAS 8006-64-2) has been tested in a study conducted according to OECD test guideline 473 and under GLP (Bohnenberger). No evidence of cytogenic activity was observed. Cytogenicity was tested using human peripheral lymphocytes. The tests were conducted with and without metabolic activation (phenobarbital and β-naphthoflavone-induced rat liver S9 mix).

In addition to these key studies, data are available for a number of constituents of TOPP none of which indicate that they would contribute a genotoxic effect.The results are summarised as follows:

α-Pinene (CAS 80-56-8): Negative in vitro (DNA damage and/ or repair, gene mutation study in bacteria) and in vivo(similar to OECD 474)

δ-3-carene (CAS13466-78-9): Negative in vitro (OECD 471)

Terpinolene/Isoterpinolene (CAS 586-62-9): Negative in vitro (OECD 471, OECD 473, OECD 487)

1,8-Cineole (CAS 470-82-6): Negative in vitro (OECD 471, chromosome aberration and sister chromatid exchange, Salmonella mutagenicity test)

Alpha-Terpineol (CAS 98-55-5): Negative in vitro (similar to OECD 471, OECD 476)

Trans-Anethole (CAS 4180-23-8): Negative in vitro (similar to OECD 482, similar to OECD 471, similar to OECD 473, DNA damage and/ or repair, mammalian cell transformation assay) and in vivo (OECD 486) and positive in in vivo mammalian germ cell study

Camphene (CAS 79-92-5):Negative in vitro (similar to OECD 471, OECD 476) and in vivo (OECD 474)

Dimethyl disulfide (CAS 624-92-0): Negative in vitro (OECD 471, OECD 473, OECD 476, OECD 482) and in vivo(OECD 474, OECD 482)

Dimethyl sulfide (CAS 75-18-3): Negative in vitro (OECD 476, OECD 471, DNA damage and/ or repair) and in vivo(similar to OECD 474)

Methanethiol (CAS 74-93-1): Negative in vivo (OECD 474)

Justification for classification or non-classification

The available information for the substance indicates that when tested in vitro, Turpentine Oil (CAS number 8006 -64 -2) does not induce mutations in bacterial or mammalian cells, nor chromosome aberrations in mammalian cells. There is no justification from in vitro results for testing in vivo. Therefore it is considered that classification for mutagenicity is not required.