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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Key study: Test method OECD 471.GLP study. The test item had no mutagenic activity in the bacterium tester strains.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 March 2014 - 10 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD 471. GLP 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:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver
Test concentrations with justification for top dose:
Initial Mutation Test:
TA98 and TA1537, -S9; TA100 and TA1535, +/- S9: 50, 15.81, 5, 1.581, 0.5, 0.1581; 0.05 and 0.0158 μg/plate
TA99 and TA1537, +S9; E coli WP2 uvrA, +/- S9: 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate
Confirmatory Mutation Test:
TA98 and TA1537, -S9; TA100 and TA1535, +/- S9: 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate
TA99 and TA1537, +S9; E coli WP2 uvrA, - S9: 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate
E coli WP2 uvrA, + S9: 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.158 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and Acetone. The test item was insoluble in Distilled water. The formulation at 100 mg/mL concentration using Acetone as vehicle was suitable for the test, while the test item was only partially soluble in DMSO at the same concentration. Therefore, Acetone was selected for vehicle (solvent) of the study.
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
Remarks:
Acetone, DMSO and distilled water
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535, -S9 (2 µg/plate)
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
TA98, -S9 (4 µg/plate)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, -S9 (50 µg/plate)
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA, -S9 (2 µg/plate)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all Salmonella strains, +S9 (2 µg/plate) and WP2 uvrA, +S9 (50 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Plate incorporation (initial mutation test):
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item (or controls) and other components (top agar, overnight culture of test strain, phosphate buffer (pH 7.4) or S9 mix) were prepared freshly and added to the overlay (45°C). This solution was mixed and poured on the surface of minimal agar plates. After preparation, the plates were incubated at 37°C for 48 hours.

Pre-incubation (confirmatory mutation test):
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. After preparation, the plates were incubated at 37°C for 48 hours.

NUMBER OF REPLICATIONS: 3 replicates per dose and controls.

EVALUATION OF EXPERIMENTAL DATA:
The colony numbers were determined by manual counting.
Visual examination of the plates was performed, precipitation or signs of growth inhibition (if any) were recorded.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated.
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.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(see below)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The observed revertant counts in the preliminary experiment were mostly in the normal range at the non-cytotoxic concentrations (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). Strong inhibitory, cytotoxic effect of the test item was detected on the plates in TA98 at 5000, 2500, 1000, 316 and 100 μg/plate +/- S9 and at 31.6 μg/plate -S9; in TA100 at 5000, 2500, 1000, 316, 100 and 31.6 μg/plate +/- S9; and in E. coli WP2 uvrA at 5000, 2500, 1000, 316 and 100 μg/plate +/- S9. No insolubility was observed on the plates in the preliminary experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Strong inhibitory, cytotoxic effect of the test item was seen in the Initial Mutation Test in TA98 and TA1537at 158.1, 50 and 15.81 μg/plate -S9 and at 500, 158.1 and 50 μg/plate +S9; in TA100 and TA1535 at 158.1, 50 and 15.81 μg/plate +/- S9; and in E. coli WP2 uvrA at 500, 158.1 and 50 μg/plate -S9 and at 500 and 158.1 μg/plate +S9. Similar effect was seen in the Confirmatory Mutation Test in TA98 and TA1537 at 50, 15.81 and 5 μg/plate -S9 and at 158.1, 50 and 15.81 μg/plate +S9; in TA100 and TA1535 at 50, 15.81 and 5 μg/plate -S9 and at 50 and 15.81 μg/plate +S9; and in E. coli WP2 uvrA at 158.1, 50 and 15.81 μg/plate -S9 and at 500, 158.1 and 50 μg/plate +S9.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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

500

Mean

--

0.0

--

--

--

--

--

0.0

0.0

0.0

MF

--

0.00

--

--

--

--

--

0.00

0.00

0.00

158.1

Mean

0.0

3.3

0.0

12.0

0.0

0.0

0.0

0.0

4.7

15.0

MF

0.00

0.14

0.00

0.12

0.00

0.00

0.00

0.00

0.15

0.40

50

Mean

2.7

18.0

5.3

47.0

0.0

4.3

1.0

2.3

17.7

27.7

MF

0.13

0.75

0.07

0.46

0.00

0.37

0.14

0.32

0.57

0.74

15.81

Mean

20.3

22.7

51.7

99.0

6.3

9.7

5.0

9.3

35.3

35.0

MF

1.02

0.94

0.64

0.97

1.46

0.83

0.71

1.27

1.14

0.94

5

Mean

21.3

27.7

86.0

98.3

8.3

11.7

5.3

10.0

38.0

42.7

MF

1.07

1.15

1.06

0.96

1.92

1.00

0.76

1.36

1.23

1.14

1.581

Mean

22.0

30.0

83.3

88.3

12.0

11.0

5.3

9.3

29.0

41.7

MF

1.10

1.25

1.02

0.87

2.77

0.94

0.76

1.27

0.94

1.12

0.5

Mean

19.7

27.3

76.3

93.0

11.3

12.0

4.0

7.3

33.7

37.0

MF

0.98

1.14

0.94

0.91

2.62

1.03

0.57

1.00

1.09

0.99

0.1581

Mean

15.7

26.7

83.0

101.3

11.0

12.3

4.3

9.3

37.3

33.3

MF

0.78

1.11

1.02

0.99

2.54

1.06

0.62

1.27

1.20

0.89

0.05

Mean

23.0

--

90.3

95.3

12.3

12.0

8.0

--

--

--

MF

1.17

--

1.11

0.93

2.85

1.03

1.14

--

--

--

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

500

Mean

--

-

--

--

--

--

--

--

--

0.3

MF

--

--

--

--

--

--

--

--

--

0.01

158.1

Mean

--

11.0

--

--

--

--

--

1.7

1.0

7.7

MF

--

0.58

--

--

--

--

--

0.25

0.03

0.21

50

Mean

5.0

22.3

8.3

62.0

0.0

8.3

0.0

6.0

1.7

12.3

MF

0.23

1.18

0.10

0.52

0.00

0.86

0.00

0.90

0.05

0.35

15.81

Mean

9.7

29.7

27.3

103.3

3.7

12.3

2.7

10.7

16.3

17.3

MF

0.44

1.56

0.32

0.86

0.52

1.28

0.62

1.60

0.52

0.49

5

Mean

15.7

28.7

57.7

110.0

8.0

14.3

4.0

7.7

21.3

38.7

MF

0.71

1.51

0.68

0.92

1.14

1.48

0.92

1.15

0.68

1.08

1.581

Mean

22.3

26.3

61.0

103.0

5.0

9.3

5.3

9.7

21.7

39.0

MF

1.02

1.39

0.72

0.86

0.71

0.97

1.23

1.45

0.69

1.09

0.5

Mean

20.7

30.3

76.0

109.0

5.3

11.0

5.3

8.3

21.7

34.0

MF

0.94

1.60

0.90

0.91

0.76

1.14

1.23

1.25

0.69

0.95

0.1581

Mean

24.3

27.0

82.0

103.7

6.0

10.0

7.3

10.3

20.0

32.7

MF

1.11

1.42

0.97

0.86

0.86

1.03

1.69

1.55

0.64

0.92

0.05

Mean

18.0

29.3

92.0

120.7

8.3

8.3

7.7

11.0

29.3

--

MF

0.82

1.54

1.09

1.01

1.19

0.86

1.77

1.65

0.94

--

0.01581

Mean

18.0

--

80.3

113.7

11.0

11.7

5.3

--

-

--

MF

0.82

--

0.95

0.95

1.57

1.21

1.23

--

--

--

In the initial mutation test, the highest revertant rate was observed in TA1535 at 0.05 μg/plate -S9 (the mutation factor value was 2.85). Higher numbers of revertant colonies compared to the Acetone control plates were detected at several concentrations in this strain. However, there was no dose response, the observed mutation factor value remained below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range. Furthermore, higher number of revertant colonies compared to the Acetone control were detected for untreated control (MF: 2.77) and Distilled water control (MF: 2.08) without metabolic activation also in this strain, indicating a higher than usual variability in this case.

In the confirmatory mutation test, the highest revertant rate was observed in TA1537 at 0.05 μg/plate concentration -S9. The observed mutation factor value was 1.77. However, no clear dose-dependent relationship was observed, and the observed mutation factor value was well below the biologically relevant threshold value. The number of revertant colonies was within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main test in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main test at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

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

The test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). Based on the results of the Solubility Test, the test item was dissolved in Acetone. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were as follows:

Initial Mutation Test:

TA98 and TA1537, -S9; TA100 and TA1535, +/- S9: 50, 15.81, 5, 1.581, 0.5, 0.1581; 0.05 and 0.0158 μg/plate

TA99 and TA1537, +S9; E coli WP2 uvrA, +/- S9: 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate

Confirmatory Mutation Test:

TA98 and TA1537, -S9; TA100 and TA1535, +/- S9: 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate

TA99 and TA1537, +S9; E coli WP2 uvrA, - S9: 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate

E coli WP2 uvrA, + S9: 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.158 μg/plate

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls and were mostly within the historical control range and were within the normal biological variability of the test system. Strong inhibitory, cytotoxic effect of the test item was detected in the Initial Mutation Test and Confirmatory Mutation Test in all examined bacterial strains with or without metabolic activation. The tests were considered to be valid. In conclusion, the test item had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.

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

Additional information

Additional information from genetic toxicity in vitro:

Key study: The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). None of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. Strong inhibitory, cytotoxic effect of the test item was detected in the Initial Mutation Test and Confirmatory Mutation Test in all examined bacterial strains with or without metabolic activation. The tests were considered to be valid. Based on these results, the test item had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.


Justification for selection of genetic toxicity endpoint
Only one study available.

Justification for classification or non-classification

Based on the available data, the substance is not classified for mutagenicity according to CLP Regulation (EC) no. 1272/2008.