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Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

Key study. Method according to OECD 471, GLP study. The test item was found to be mutagenic for the referenced strains Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 and in Escherichia coli WP2 (uvrA), with and without metabolic activation.

Key study. Method equivalent to OECD 471, GLP study. The test item was found to be mutagenic for the referenced strains Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 (uvr A), with and without metabolic activation.

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
Study period:
From November 24th to November 26th, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine biosynthethic genes.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix containing rat liver homogenate from phenobarbital and B-naphthoflavone induced rats
Test concentrations with justification for top dose:
0, 5, 15.81, 50, 158.1, 500, 1581, 5000 µg/plate.
The top dose is the recommended maximum test concentration for soluble non-cytotoxic substances in OECD 471 (5 mg/plate), based on the preliminary range-finding test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: According to the available information the test item degrades in contact with water. The Ames test media needs to contain water. In the main tests, the test item was added to media as a DMSO solution, in order to minimise the possibility of degradation before interaction with the test organism bacteria.
Untreated negative controls:
yes
Remarks:
untreated control.
Negative solvent / vehicle controls:
yes
Remarks:
distilled water.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
DMSO as vehicle. S. typhimurium TA98
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
sodium azide
Remarks:
Distilled water as vehicle. S. typhimurium TA100; TA1535
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
DMSO as vehicle. S. typhimurium TA1537
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
Distilled water as vehicle. E. coli WP2 uvrA
Positive controls:
yes
Remarks:
With metabolic activation.
Positive control substance:
other: 2-aminoanthracene
Remarks:
DMSO as vehicle. All Salmonella strains and E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate. In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the plate incorporation method (Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2 uvrA) as well as the pre-incubation method (Salmonella typhimurium TA98, TA1537) was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of metabolic activation system (±S9 mix). Each test was performed with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.

DURATION
- Preincubation period: 20 minutes with the pre-incubation method.
- Exposure duration: 48 hours.
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): Histidine-free medium.

NUMBER OF REPLICATIONS: Each tested sample in triplicate.

DETERMINATION OF CYTOTOXICITY: Yes
- Method: The revertant colony numbers and the inhibition of background lawn of auxotrophic cells.

OTHER:
An initial mutation test and a confirmatory mutation test were conducted.
Depending on the results for reach tester strain, the plate incorporation or the preincubation method were used at the confirmatory mutation test.
The plate incorporation method was used in the confirmatory mutation test when positive responses were recorded.
The preincubatin method was used in the confirmatory mutation test when no-positive responses were observed.
Rationale for test conditions:
The appropriate vehicle and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. The test item was soluble at 100 mg/ml concentration in Distilled water* or in DMSO or in DMF. Due to the better biocompatibility to the test system, Distilled water was selected as vehicle (solvent) for the Preliminary Test. However additional research and discussions were made and it was recommended to test the test item using DMSO as vehicle in the main tests in order to obtain a better assessment of the mutagenicity of the test item since this might be more similar to the situation of the possible human exposure.
Evaluation criteria:
Criteria for a Positive Response:
A test item was considered mutagenic if:
-a dose–related increase in the number of revertants occurred and/or;
-a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

DMSO was used as vehicle in order to ensure a maximal concentration of the test item.

RANGE-FINDING/SCREENING STUDIES: The formulation with distilled water looked opalescent in the first but became clear solution within a half minut. During the reaction warming was observed.
The initial range finding test was conducted with 2 tester strains: S. typhimurium TA98 and TA 100 with the following concentrations: 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: Sporadically, lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Test. However, the mean numbers of revertant colonies were in the historical control range in all cases and were considered as biological variability of the test system

Table 5: Summary Table of the Initial Mutation Test

Concentrations
(µg/plate)

Mean
values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

17.3

24.3

121.7

105.3

29.0

17.0

4.7

6.7

27.0

29.3

MF

0.83

1.04

1.13

1.03

1.01

1.55

0.88

1.00

0.89

1.10

DMSO
control

Mean

21.0

23.3

107.7

102.0

28.7

11.0

5.3

6.7

30.3

26.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

105.0

--

25.7

--

--

--

29.0

--

MF

--

--

0.98

--

0.90

--

--

--

0.96

--

5000

Mean

23.3

54.0

249.0

363.3

96.7

193.7

9.0

6.7

70.0

173.3

MF

1.11

2.31

2.31

3.56

3.37

17.61

1.69

1.00

2.31

6.50

1581

Mean

28.3

28.3

136.3

142.0

64.7

149.7

7.7

6.7

57.7

71.0

MF

1.35

1.21

1.27

1.39

2.26

13.61

1.44

1.00

1.90

2.66

500

Mean

23.3

28.7

117.3

132.3

29.0

43.7

6.0

9.7

33.7

56.3

MF

1.11

1.23

1.09

1.30

1.01

3.97

1.13

1.45

1.11

2.11

158.1

Mean

23.3

27.0

97.0

85.7

18.7

27.7

2.3

9.0

37.3

42.3

MF

1.11

1.16

0.90

0.84

0.65

2.52

0.44

1.35

1.23

1.59

50

Mean

18.3

20.3

97.3

99.7

19.7

17.3

5.3

9.3

30.3

39.3

MF

0.87

0.87

0.90

0.98

0.69

1.58

1.00

1.40

1.00

1.48

15.81

Mean

20.3

26.0

100.7

89.7

16.3

12.7

9.0

6.3

33.0

39.3

MF

0.97

1.11

0.93

0.88

0.57

1.15

1.69

0.95

1.09

1.48

5

Mean

21.0

22.7

94.0

86.7

13.3

15.3

6.7

8.3

30.3

42.3

MF

1.00

0.97

0.87

0.85

0.47

1.39

1.25

1.25

1.00

1.59

NPD (4mg)

Mean

316.0

--

--

--

--

--

--

--

--

--

MF

15.05

--

--

--

--

--

--

--

--

--

2AA (2mg)

Mean

--

2241.3

--

2149.3

--

220.0

--

217.3

--

--

MF

--

96.06

--

21.07

--

20.00

--

32.60

--

--

2AA (50mg)

Mean

--

--

--

--

--

--

--

--

--

176.0

MF

--

--

--

--

--

--

--

--

--

6.60

SAZ (2mg)

Mean

--

--

880.0

--

1160.0

--

--

--

--

--

MF

--

--

8.38

--

45.19

--

--

--

--

--

9AA (50mg)

Mean

--

--

--

--

--

--

412.7

--

--

--

MF

--

--

--

--

--

--

77.38

--

--

--

MMS (2mL)

Mean

--

--

--

--

--

--

--

--

886.7

--

MF

--

--

--

--

--

--

--

--

29.89

--

--: Not applicable

Table 6:Summary Table of the Confirmatory Mutation Test

Concentrations
(µg/plate)

Mean
values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

15.0

30.3

93.0

86.3

11.3

10.0

3.3

4.7

22.7

25.0

MF

0.73

1.42

1.04

0.95

0.89

1.15

1.25

0.67

1.03

1.06

DMSO
control

Mean

20.7

21.3

89.0

90.7

12.7

8.7

2.7

7.0

22.0

23.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

87.3

--

12.7

--

--

--

23.0

--

MF

--

--

0.98

--

1.00

--

--

--

1.05

--

5000

Mean

98.3

108.3

245.0

206.3

117.0

51.7

23.0

31.3

82.3

292.3

MF

4.76

5.08

2.75

2.28

9.24

5.96

8.63

4.48

3.74

12.35

1581

Mean

58.3

65.3

124.0

115.3

38.3

79.0

10.0

16.0

74.7

199.3

MF

2.82

3.06

1.39

1.27

3.03

9.12

3.75

2.29

3.39

8.42

500

Mean

31.0

32.7

80.7

108.7

19.3

24.0

5.0

7.7

59.7

103.3

MF

1.50

1.53

0.91

1.20

1.53

2.77

1.88

1.10

2.71

4.37

158.1

Mean

22.3

31.3

89.0

90.0

13.7

14.3

9.0

6.7

37.7

39.3

MF

1.08

1.47

1.00

0.99

1.08

1.65

3.38

0.95

1.71

1.66

50

Mean

23.0

26.0

81.3

83.0

11.3

12.0

5.7

7.3

29.3

28.3

MF

1.11

1.22

0.91

0.92

0.89

1.38

2.13

1.05

1.33

1.20

15.81

Mean

19.7

29.0

79.0

96.3

14.7

16.3

6.0

7.7

28.0

31.0

MF

0.95

1.36

0.89

1.06

1.16

1.88

2.25

1.10

1.27

1.31

5

Mean

15.7

28.0

74.7

80.0

11.0

10.7

9.0

6.7

19.7

26.0

MF

0.76

1.31

0.84

0.88

0.87

1.23

3.38

0.95

0.89

1.10

NPD (4mg)

Mean

333.3

--

--

--

--

--

--

--

--

--

MF

16.13

--

--

--

--

--

--

--

--

--

2AA (2mg)

Mean

--

2373.3

--

2272.0

--

228.0

--

209.3

--

--

MF

--

111.25

--

25.06

--

26.31

--

29.90

--

--

2AA (50mg)

Mean

--

--

--

--

--

--

--

--

--

176.0

MF

--

--

--

--

--

--

--

--

--

7.44

SAZ (2mg)

Mean

--

--

1146.7

--

1114.7

--

--

--

--

--

MF

--

--

13.13

--

88.00

--

--

--

--

--

9AA (50mg)

Mean

--

--

--

--

--

--

392.0

--

--

--

MF

--

--

--

--

--

--

147.00

--

--

--

MMS (2mL)

Mean

--

--

--

--

--

--

--

--

1082.7

--

MF

--

--

--

--

--

--

--

--

47.07

--

--: Not applicable

Conclusions:
The test item was found to be mutagenic for the referenced strains Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 and in Escherichia coli WP2 (uvrA), with and without metabolic activation.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay, according to OECD 471 (GLP study). The assay was performed in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 and in Escherichia coli WP2 (uvrA), in the presence and in the absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats. In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the plate incorporation method (Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2 uvrA) as well as the pre-incubation method (Salmonella typhimurium TA98, TA1537) was used. Each test was performed with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate. The concentrations tested were 0, 5, 15.81, 50, 158.1, 500, 1581, 5000 µg/plate of test item.

Under test conditions, the test item had mutagenic activity on all tester strains. Therefore, it is considered to be mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 6th, to November 15th, 2007.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH. Topic S2A Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH. Topic S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: C0014.
- Expiration date of the lot/batch: 31 July 2008.
- Purity: 99.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8ºC) in the dark.
- Stability under test conditions: stable.
Target gene:
histidine-requring gene for all Samlmonella typhimurium strains, tryptophan-requiring gene for E. coli uvr A.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix.
Test concentrations with justification for top dose:
100, 333, 1000, 3330 and 5000 µg/plate, based on the results of the dose range-finding test (at concentrations 3-5000µg/plate): the bacterial background lawn was not reduced at any of the concentrations tested, and no decrease in the number of revertants was observed.
The top dose is the recommended maximum test concentration for soluble non-cytotoxic substances in OECD 471 (5 mg/plate), based on the preliminary range-finding test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (SeccoSov, Merck, Darmstadt, Germany).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene for all strains with metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Dose range finding test: Eight concentrations 3, 10, 33, 100, 333, 1000, 3330, and 5000 µg/plate were tested in triplicated, both with and without S9-mix.
- Mutation assay: Five concentrations in the range 100-5000 µg/plate were tested in triplicate, both with and without S9-mix. Top agar in top agar tubes was molten and heated to 45ºC. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in DMSO and either 0.5ml S9-mix (activation assays) or 0.5 ml 0.1M phosphate buffer (non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0ºC for 48h. After this period, revertant colonies were counted manually (if less than 40 colonies per plate were present or if the test item precipitated) or automatically (if more than 40 colonies were present), with a Biocount 4000 Pro-S-colony counter.

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial backgroun lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed in the presence and absence of S9-mix.
Rationale for test conditions:
The appropriate vehicle and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test.
Evaluation criteria:
A test substance is considered positive in the test if the number of revertants is greater than two (for TA100) or three (for all other strains) times the concurrent control, and if the positive response is reproducible in at least one independently repeated experiment.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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 and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Toxicity: there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the presence and absence of S9 mix.

- Mutagenicity: In tester strain TA1535, the test item induced up to 20-fold dose related, increases in the number of revertant colonies compared to the solven control both in the absence and presence of S9 mix. In tester strain TA98, it induced up to 2.8- and 2.1-fold dose related, increases in the number of revertant colonies compared to the solven control both in the absence and presence of S9 mix. In tester strain TA 1537, no increase in the number of revertant colonies was observed.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: In the tester strain TA100, the test item induced up to 8.8 and 6.6-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In tester strain WP2uvrA, it induced up to 28- and 27-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- The negative and strain-specific positive control values were within the laboratory's historical control data ranges, indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Table 1. Mutagenic response of EMCA in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain.

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Without S9-mix

Positive control

1025 ± 30

411 ± 61

1143 ± 29

954 ± 53

1047 ± 54

Solvent control

10 ± 1

6 ± 2

14 ± 2

88 ± 3

15 ± 7

3

 

 

 

94 ± 7

16 ± 3

10

 

 

 

102 ± 13

17 ± 6

33

 

 

 

102 ± 10

17 ± 6

100

14 ± 3

8 ± 2

18 ± 6

110 ± 8

25 ± 2

333

21 ± 4

6 ± 0

16 ± 4

152 ± 10

42 ± 12

1000

55 ± 8

10 ± 1

19 ± 6

271 ± 14

80 ± 20

3330

165 ± 23

7 ± 1

39 ± 7

592 ± 128

277 ± 32

5000

202 ± 13

6 ± 3

37 ± 2

776 ± 62

416 ± 49

With S9-mix

Positive control

279 ± 2

328 ± 45

740 ± 129

811 ± 129

280 ± 45

Solvent control

9 ± 2

6 ± 2

22 ± 7

79 ± 11

14 ± 2

3

 

 

 

82 ± 5

17 ± 3

10

 

 

 

77 ± 7

14 ± 4

33

 

 

 

98 ± 5

23 ± 4

100

10 ± 2

5 ± 2

23 ± 3

95 ± 7

27 ± 6

333

22 ± 2

7 ± 1

24 ± 3

129 ± 13

49 ± 9

1000

50 ± 19

7 ± 2

26 ± 4

255 ± 17

98 ± 20

3330

102 ± 33

5 ± 1

44 ± 8

421 ± 86

302 ± 22

5000

182 ± 49

8 ± 2

46 ± 3

523 ± 104

371 ± 135

Solvent control: 0.1 ml DMSO.

Conclusions:
The test item was found to be mutagenic for the referenced strains Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 (uvr A), with and without metabolic activation.
Executive summary:

Evaluation of the mutagenic activity of the test item was performed by a method equivalent to OECD 471 (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and a tryptophan-requiring strain of Escherichia coli (WP2 uvr A) were tested using the plate incorporation method, in the presence and absence of S9 -mix metabolic activation system (rat liver S9 induced by a combination of phenobarbital and b-naphtoflavone). For the preliminary test, eight concentrations (3, 10, 33, 100, 333, 1000, 3330, and 5000 µg/plate) were tested in triplicated, both with and without S9-mix. In the main test, five concentrations (100, 333, 1000, 3330 and 5000 µg/plate) were tested, with and without metabolic activation.

Under test conditions, the test item induced dose related increases in the number of revertant colonies in four tester strains (TA1535, TA98, TA100 and WP2 uvrA), with and without metabolic activation. Therefore, the test item is considered to be mutagenic.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro:

Key study. The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay, according to OECD 471 (GLP study). The assay was performed in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 and in Escherichia coli WP2 (uvrA), in the presence and in the absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats. Under test conditions, the test item had mutagenic activity on all tester strains. Therefore, it is considered to be mutagenic.

Key study. The test item was tested for potential mutagenic activity by a method equivalent to OECD 471 (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and a tryptophan-requiring strain of Escherichia coli (WP2 uvr A) were tested, in the presence and absence of S9 -mix metabolic activation system (rat liver S9 induced by a combination of phenobarbital and b-naphtoflavone).

Under test conditions, the test item induced dose related increases in the number of revertant colonies in four tester strains (TA1535, TA98, TA100 and WP2 uvrA), with and without metabolic activation. Therefore, the test item is considered to be mutagenic.

Justification for classification or non-classification

There is not enough evidence for the classification of the test item as mutagenic. Accordingly, the test substance is not classified in accordance with CLP Regulation (EC) No. 1272/2008.