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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to generally valid and/or internationally accepted testing guidelines.

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dihydromyrcenol
- Physical state: Clear, colorless slightly viscous liquid
- Analytical purity: 99.2% (COA)
- Purity test date: 29 October 2010
- Lot/batch No.: 9SFD04
- Expiration date of the lot/batch: 27 October 2011
- Storage condition of test material: Room temperature in the dark under nitrogen
- Other: Information relating to the identity, purity and stability of the test material was the responsibility of the Sponsor

Method

Target gene:
Salmonella typhimurium:
TA 1535, TA 100: hisG46
TA 1537: hisC3076
TA 98: hisD3052
Escherichia coli (WP2uvrA/pKM101):
contains ochre mutation; deficient in DNA repair system 9uvrA); contains the pKM101 plasmid
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate

Experiment 1 (Range-finding)
15, 50, 150, 500, 1500 and 5000 microg/plate

Mutation Test (Main Test) - with preincubation
TA100 (with/without S9), TA1535 and TA1537 (without S9) and E. coli (without S9): 1.5, 5, 15, 50, 150, 500 and 1500 microg/plate

TA98 (with/without S9), TA1535 and TA1537 (with S9) and E. coli (with S9): 15, 50, 150, 500, 1500 and 5000 microg/plate
Vehicle / solvent:
dimethyl sulphoxide

The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Pos. Control for TA98 at 5 microg/plate (+S9)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
Pos. Control for TA100 at 1 microg/plate (+S9); TA1535 and TA1537, 2 microg/plate (+S9); and E. coli WP2uvrA- at 10 microg/plate (+S9)
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Pos. Control for E. coli WP2uvrA- at 2 microg/plate (-S9); TA100 at 3 microg/plate (-S9); TA1535 at 5 microg/plate (-S9)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Pos. Control for TA1537 at 80 microg/plate (-S9)
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Pos. Control for TA98 at 0.2 microg/plate (-S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
A standard plate incorporation method was employed.

Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test were conducted in the presence or absence of S9. Ten dose levels and controls were tested up to and including 5,000 microg/plate at approximately half-log intervals. The assay was conducted by mixing 0.1 ml the bacterial culture (TA100 or WP2uvrA-), and 0.1 ml of the vehicle or test chemical mixture, 0.5 mL of S9-mix or phosphate buffer and 2.0 ml of molten agar supplemented with trace histidine or tryptophan and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 deg C, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Experiment 1 (Range-finding Test):
Six concentrations of the test material were assayed with or without S9-mix against each tester strain using the above described direct plate incorporation method. An additional dose level and expanded dose range were selected in order to achieve both four non-toxic doses and the toxic limit of the test material.

Experiment 2 (Main Test):
A second experiment was performed with fresh bacterial cultures, test material and control solutions. The dose ranges selected were based on the range-finding test and an aborted main test (data not shown). Preincubation was employed. Thus, measured aliquots (0.1 mL) of each bacterial culture were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of vehicle or test material formulation and incubated for 20 minutes at 37 deg C with shaking at approximately 130 rpm prior to addition of 2 mL of molten trace histidine or tryptophan supplemented top agar. The contents of the tubes were mixed and poured onto the surface of Vogel-Bonner Minimal agar plates. This procedure was repeated for each bacterial strain either with or without S9.

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.
Evaluation criteria:
Acceptance Criteria:

The following criteria must be met for acceptance:
- All tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls.
- The appropriate characteristics of each tester strain must be confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor.
- All tester strain cultures should be in the range of 1 to 9.9 x 10^9 bacteria per mL.
- Each mean positive control value should be at least 2x the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains and the integrity of the S9-mix
- The test should include a minimum of four non-toxic dose levels.

Evaluation Criteria:

A dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain either with or without metabolic activation. Biological relevance of the response is to be considered first, as recommended by the UKEMS sub-committee on Guidelines for Mutagenicity Testing (1989). Statistical methods can be used as an aid to evaluation but may not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Mean values with standard deviations were reported.

Results and discussion

Test resultsopen allclose all
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Please refer to Tables 1 to 5 for details of results.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was toxic at 5000 microg/plate to tester strains TA100 and WP2uvrA-. The results are summarized in Table 1.

Table 1: Numbers of revertant colonies in the preliminary toxicity test

With (+) or without (-) S9-mix

Strain

Dose (mg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

93

100

120

101

123

133

132

106

128

152

113*

+

TA100

62

107

77

82

81

88

94

84

84

74

0*

-

WP2uvrA-

18

15

16

18

22

33

24

14

19

20

17*

+

WP2uvrA-

28

32

22

27

22

20

24

25

25

31

15*

* Partial absence of bacterial background lawn

Table 2: Range-finding test without metabolic activation

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

101

24

18

17

11

15

81

30

19

15

10

50

86

28

19

19

10

150

91

23

17

20

9

500

99

27

18

18

11

1500

92

27

20

14

10

5000

82*

15*

13

12*

2*

ENNG (3)

421

ENNG (5)

907

ENNG (2)

596

4NQO (0.2)

167

9AA (80)

354

* Partial absence of background lawn in all replicate plates.

Abreviations: ENNG, N-ethyl-Nā€™-nitro-N-nitrosoguanidine; 9AA, 2-aminoacridine; BP, benzo(a)pyrene; 2AA, 2-aminoanthracene; 4NQO, 4-nitroquinoline-1-oxide

Table 3: Range-finding test with metabolic activation

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

88

14

22

19

14

15

90

15

22

21

14

50

87

14

21

15

14

150

88

10

19

21

10

500

74

11

23

19

10

1500

84

9

17

16

9

5000

72*

2*

17

12*

3*

2AA (1)

760

2AA (2)

212

195

2AA (10)

244

BP (5)

119

* Partial absence of background lawn in all replicate plates.

Table 4: Main Test without metabolic activation

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

106

28

26

11

12

1.5

107

24

26

N/T

14

5

103

27

17

N/T

12

15

108

29

20

15

9

50

100

25

21

10

8

150

107*

28

26

15

9

500

76*

27*

25

10

8*

1500

0*

0*

0*

0*

0*

5000

N/T

N/T

N/T

0*

N/T

ENNG (3)

406

ENNG (5)

291

ENNG (2)

498

4NQO (0.2)

100

9AA (80)

937

* Partial absence of background lawn in all replicate plates.

N/T - not tested at this dose level.

Table 5: Main Test with metabolic activation

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

114

10

18

19

9

1.5

102

N/T

N/T

N/T

N/T

5

115

N/T

N/T

N/T

N/T

15

90

11

17

22

11

50

102

11

21

25

8

150

103

14

19

20

10

500

95*

11

15

18

7

1500

0*

0*

0*

0*

0*

5000

N/T

0*

0*

0*

0*

2AA (1)

813

2AA (2)

230

199

2AA (10)

212

BP (5)

248

* Partial absence of background lawn in all replicate plates.

N/T - not tested at this dose level.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was considered non-mutagenic under the conditions of this test.
Executive summary:

In the first experiment, the test material caused a visible reduction in the growth f the bacterial background lawns of all the Salmonella stains at 5000 microg/plate in both the presence and absence of S9. No toxic response to Escherichia coli strain WP2uvrA- was noted. In the second experiment (with pre-incubation) the test material reduced the bacterial background lawns of all the tester strains in both the presence and absence of S9. The sensitivity of the various stains varied with the presence or absence of S9 and the strain involved; therefore, the test material was tested up to the maximum recommended dose level. No test material precipitate was observed on the plates at any of the doses tested either with or without S9.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains.

The test material was considered to be non-mutagenic under the conditions of this test.