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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and TA 102 with and without metabolic activation

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May - 01 Jul 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(Commission Regulation (EC) No 440/2008 of 30 May 2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, U.K.
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix) prepared from rats treated with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation in TA 100
Experiment 1: 15, 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation in all strains
Experiment 2: 5, 15, 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation in all strains
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: In solubility checks the test material was fully soluble in DMSO at 50 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene (2AA), 1,8-Dihydroxyanthraquinone (DAN)
Details on test system and experimental conditions:
METHOD OF APPLICATION: experiment 1: plate incubation method; experiment 2: pre-incubation method for the test groups and vehicle controls, positive and negative controls were dosed using standard plate incubation method

DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: clearing of the bacterial background lawn
Evaluation criteria:
There are several criteria for determining a positive result, such as 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 with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by UKEMS (Kirkland ( 1989) Sub-committee on Guidelines for mutagenicity Testing, Report-Part III, Cambridge University Press) can also be used as an aid to evaluation, however, statistical significance will 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.

Acceptance criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
Spontaneous revertants of tester strain cultures for vehicle and negative control should be in the range of historical control data. The appropriate characteristics for each tester strain have been confirmed (eg rfs cell-wall-mutation, pKM101 plasmid R-factor etc.). All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per mL. Each mean positive control value should be at least two times the respective vehicle control for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and integrity of the S9-mix. There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.
Statistics:
Mean value and standard deviations were calculated.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: +/-S9: ≥ 1500 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: -S9: ≥ 1500 µg/plate, +S9: 5000 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: +/-S9: ≥ 1500 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: +/-S9: ≥ 1500 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: +/-S9: 5000 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitate was observed on the plates at any concentrations tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for use of the maine test, a preliminary test was carried out to determine the toxicity of the test material. Ten doses of the test material and a vehicle control (DMSO) were tested in TA 100 using the plate incubation method. The test material was initially toxic at and above 1500 µg/plate.

HISTORICAL CONTROL DATA: Positive and vehicle controls were within the normal range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1 the test substance caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains both in absence and presence of S9-mix, initially from 1500 µg/plate. In Experiment 2 the test material exhibited toxicity as weakened background lawns to all of the bacterial strains, initially from 500 µg/plate and 1500 µg/plate in the absence and presence of S9-mix, respectively.

Table 1. Test results of experiment 1 (plate incorporation method)

EXPERIMENT 1

S9-Mix

Without

 

Test substance (µg/plate)

TA 100

TA 1535

TA 102

TA 98

TA 1537

SC

77 ± 8.5

11 ± 0.6

237 ± 16.7

23 ± 4.6

15 ± 8.3

15

80 ± 5.8

10 ± 0.6

232 ± 27.2

23 ± 1.7

15 ± 0.6

50

79 ± 10

11 ± 2.6

240 ± 30.1

17 ± 5.9

13 ± 5.5

150

71 ± 2.3

10 ± 1.5

225 ± 11.7

22 ± 8.7

13 ± 3.2

500

89 ± 6.1

14 ± 2.6

229 ± 10.5

18 ± 4.6

9 ± 4.4

1500

74 ± 13.3 S

8 ± 3.6 S

197 ± 12.6

7 ± 2.1 S

5 ± 2.3 S

5000

0 ± 0.0 T

0 ± 0.0 T

30 ± 17.4 S

0 ± 0.0 T

0 ± 0.0 T

ENNG

386 ± 47.9

103 ± 10.0

 

 

 

MMC

 

 

933 ± 199.2

 

 

4NQO

 

 

 

155 ± 12.1

 

9AA

 

 

 

 

1003 ± 210.7

S9-Mix

With

 

Test substance (µg/plate)

TA 100

TA 1535

TA 102

TA 98

TA 1537

SC

85 ± 0.6

11 ± 0.6

261 ± 23.6

23 ± 9.9

 10 ± 1.0

15

75 ± 10.1

10 ± 1.7

283 ± 6.2

26 ± 7.2

 15 ± 4.2

50

86 ± 3.2

10 ± 1.2

258 ± 27.6

25 ± 7.1

8 ± 3.6

150

78 ± 6.0

9 ± 1.0

244 ± 31.6

31 ± 1.5

11 ± 2.1

500

77 ± 2.9

8 ± 0.0

249 ± 9.8

28 ± 2.6

14 ± 2.9

1500

53 ± 4.4 S

8 ± 1.7 S

37 ± 12.7 S

21 ± 3.8 S

7 ± 6.4

5000

0 ± 0.0 T

0 ± 0.0 T

 

0 ± 0.0 T

0 ± 0.0 T

2AA

1994 ± 80.3

92 ± 7.9

 

 

148 ± 31.5

DAN

 

 

916 ± 34.2

 

 

BP

 

 

 

279 ± 36.4

 

SC = Solvent Control (DMSO)

S = Sparse bacterial background lawn

T = Toxic, no bacterial background lawn

Positive Controls: ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine; MMC: Mitomycin C; 4NQO: 4-Nitroquinoline-1-oxide; 9-AA: 9-aminoacridine; 2AA: 2-Aminoanthracene; DAN: 1,8-Dihydroxyanthraquinone; BP: Benzo(a)pyrene

 

Table 2. Test results of experiment 2 (pre-incubation method)

EXPERIMENT 2

S9-Mix

Without

 

Test

substance

(µg/plate)

TA 100

TA 1535

TA 102

TA 98

TA 1537

SC

84 ± 7.4

16 ± 3.1

222 ± 16.7

15 ± 2.9

12 ± 3.8

5

77 ± 9.5

15 ± 1.0

237 ± 4.5

16 ± 4.5

8 ± 1.5

15

70 ± 6.9

16 ± 2.6

230 ± 9.3

18 ± 1.2

8 ± 2.5

50

69 ± 15.6

18 ± 1.5

230 ± 8.4

17 ± 2.5

9 ± 4.5

150

61 ± 4.5

18 ± 1.2

208 ± 7.1

16 ± 2.1

12 ± 3.5

500

0 ± 0.0 V

14 ± 5.2 S

118 ± 8.5 S

0 ± 0.0 V

0 ± 0.0 V

1500

0 ± 0.0 T

0 ± 0.0 T

68 ± 39.2 S

0 ± 0.0 T

0 ± 0.0 T

5000

0 ± 0.0 T

0 ± 0.0 T

0 ± 0.0 T

0 ± 0.0 T

0 ± 0.0 T

ENNG

466 ± 47.0

284 ± 67.3

 

 

 

MMC

 

 

699 ± 12.1

 

 

4NQO

 

 

 

91 ± 4.7

 

9AA

 

 

 

 

1109 ± 144.2

S9-Mix

With

 

Test

substance

(µg/plate)

TA 100

TA 1535

TA 102

TA 98

TA 1537

SC

77 ± 10.5

12 ± 1.7

244 ± 14.2

22 ± 6.9

15 ± 4.4

5

73 ± 9.7

8 ± 2.5

255 ± 8.6

19 ± 4.6

11 ± 0.6

15

66 ± 16.5

15 ± 4.6

264 ± 13.3

24 ± 4.6

9 ± 5.2

50

71 ± 3.8

9 ± 2.0

266 ± 4.5

21 ± 4.5

9 ± 2.5

150

76 ± 13.3

13 ± 4.2

281 ± 11.1

27 ± 9.3

8 ± 4.4

500

69 ± 10.5

10 ± 1.0

262 ± 17.6

24 ± 6.4

9 ± 4.5

1500

0 ± 0.0 V

0 ± 0.0 T

199 ± 1.2

0 ± 0.0 V

4 ± 1.5 V

5000

0 ± 0.0 T

0 ± 0.0 T

0 ± 0.0 V

0 ± 0.0 T

0 ± 0.0 T

2AA

2559 ± 78.3

286 ± 38.7

 

 

246 ± 16.4

DAN

 

 

1532 ± 138.5

 

 

BP

 

 

 

680 ± 9.8

 

SC = Solvent Control (DMSO)

S = Sparse bacterial background lawn

T = Toxic, no bacterial background lawn

V = Very weak bacterial background lawn

Positive Controls: ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine; MMC:Mitomycin C; 4NQO: 4-Nitroquinoline-1-oxide; 9-AA: 9-aminoacridine; 2AA: 2-Aminoanthracene; DAN: 1,8-Dihydroxyanthraquinone; BP: Benzo(a)pyrene

 

 

 

 

Conclusions:
Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation up to the limit dose of 5000 µg/plate.
Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2009). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation up to 5000 µg/plate.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2009). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation up to 5000 µg/plate.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008.