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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
Study period:
From November 24th to November 26th, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

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
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
479-390-3
EC Name:
-
Cas Number:
42252-34-6
Molecular formula:
C4H8ClNO
IUPAC Name:
N-ethyl-N-methylcarbamoyl chloride
Test material form:
liquid
Remarks:
Colourless liquid with precipitation particles

Method

Target gene:
Histidine biosynthethic genes.
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
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.
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.