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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:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA (TSCA) OPPTS harmonised guidelines
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Turpentine, oil
EC Number:
232-350-7
EC Name:
Turpentine, oil
Cas Number:
8006-64-2
Molecular formula:
UVCB substance molecular formula varied but mainly C10H16, C15H24, C2H6S1, C1H4S1 and C10H18O1
IUPAC Name:
Turpentine Oil from Pulping Processes (TOPP) is a volatile oil extracted from various tree species. It consists of terpenes, mainly bicyclic monoterpenes such as alpha- and beta-pinene and delta-3-carene, and lower concentrations of monocyclic monoterpenes.

Method

Target gene:
histidine (Salmonella); tryptophan (E. coli)
Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test results
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.