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Toxicological information

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:
14.07.2015 - 15.10.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate (experiment 2); top dose chosen following the pre-experiment (experiment 1)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Negative controls:
yes
Remarks:
Medium
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with metabolic activation
Negative controls:
yes
Remarks:
Medium
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535; TA 100 without metabolic activation
Negative controls:
yes
Remarks:
Medium
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA 1537, TA 98 without metabolic activation
Negative controls:
yes
Remarks:
Medium
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA without metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in DMSO

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: spontaneous reversion rates
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

Experiment 1

Without metabolic activation

 

Dose/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

DMSO

 

9±2

11±1

24±5

140±3

39±5

Untreated

 

11 ± 2

8 ± 1

27 ± 2

160 ± 10

44 ± 5

Test item

3 µg

8 ± 2

11 ± 2

24 ± 6

135 ± 5

40 ± 3

 

10 µg

11 ± 3

12 ± 2

24 ± 3

158 ± 11

39 ± 2

 

33 µg

10 ± 5

11 ± 3

19 ± 5

139 ± 13

43 ± 8

 

100 µg

8 ± 1

10 ± 2

18 ± 4

153 ± 9

38 ± 8

 

333 µg

9 ± 3

9 ± 3

21 ± 2

126 ± 5

42 ± 7

 

1000 µg

9 ± 1

11 ± 4

27 ± 6

127 ± 6

40 ± 3

 

2500 µg

7 ± 2

5 ± 2

20 ± 2

70 ± 4

38 ± 11

 

5000 µg

7 ± 1

5 ± 2

18 ± 4

46 ± 7*

28 ± 4

NaN3

10 µg

919 ± 66

 

 

2106 ± 122

 

4-NOPD

10 µg

 

 

413 ± 9

 

 

4-NOPD

50 µg

 

90 ±5

 

 

 

MMS

2.0 µL

 

 

 

 

783 ± 68

 

With metabolic activation

 

Dose/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

DMSO

 

10 ± 2

11 ± 4

34 ± 8

117 ± 10

46 ± 8

Untreated

 

14 ± 4

9 ± 4

36 ± 2

144 ± 10

48 ± 10

Test item

3 µg

11 ± 3

12 ± 4

34 ± 5

115 ± 11

46 ± 7

 

10 µg

9 ± 1

12 ± 5

35 ± 3

107 ± 10

51 ± 7

 

33 µg

9 ± 1

12 ± 4

28 ± 6

110 ± 15

46 ± 6

 

100 µg

9 ± 1

13 ± 4

31 ± 4

116 ± 15

45 ± 4

 

333 µg

11 ± 2

12 ± 3

29 ± 3

101 ± 4

43 ± 2

 

1000 µg

8 ± 2

11 ± 2

34 ± 1

113 ± 6

43 ± 4

 

2500 µg

10 ± 3

9 ± 3

33 ± 2

86 ± 9

37 ± 7

 

5000 µg

9 ± 1

9 ± 2

26 ± 2

63 ± 11

44 ± 1

2-AA

2.5 µg

399 ± 8

225 ± 34

4168 ± 319

3201 ± 160

 

2-AA

10 µg

 

 

 

 

370 ± 37

 

Experiment 2

Without metabolic activation

 

Dose/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

DMSO

 

11 ± 2

8 ± 3

22 ± 4

176 ± 13

34 ± 4

Untreated

 

11 ± 3

11 ± 2

32 ± 14

189 ± 23

40 ± 6

Test item

3 µg

12 ± 2

7 ± 1

23 ± 4

159 ± 12

35 ± 4

 

10 µg

10 ± 4

7 ± 1

22 ± 4

175 ± 23

36 ± 2

 

33 µg

10 ± 4

6 ± 1

26 ± 4

171 ± 20

33 ± 4

 

100 µg

10 ± 4

10 ± 1

24 ± 8

164 ± 6

34 ± 7

 

333 µg

9 ± 4

8 ± 1

22 ± 3

131 ± 14

37 ± 8

 

1000 µg

8 ± 2

7 ± 2

21 ± 5

91 ± 13

28 ± 4

 

2500 µg

6 ± 1*

7 ± 2

27 ± 1

72 ± 11

27 ± 1*

 

5000 µg

4 ± 2*

7 ± 2*

5 ± 2*

0 ± 0

22 ± 2*

NaN3

10 µg

1173 ± 24

 

 

1895 ± 196

 

4-NOPD

10 µg

 

 

428 ± 28

 

 

4-NOPD

50 µg

 

103 ± 19

 

 

 

MMS

2.0 µL

 

 

 

 

580 ± 90

 

With metabolic activation

 

Dose/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

DMSO

 

9 ± 1

10 ± 3

28 ± 2

146 ± 14

43 ± 7

Untreated

 

12 ± 3

12 ± 2

40 ± 9

212 ± 8

49 ± 8

Test item

3 µg

9 ± 3

12 ± 2

31 ± 3

143 ± 6

44 ± 6

 

10 µg

10 ± 4

9 ± 5

33 ± 9

151 ± 40

47 ± 14

 

33 µg

13 ± 1

9 ± 4

33 ± 2

148 ± 23

50 ± 11

 

100 µg

9 ± 2

10 ± 4

29 ± 3

139 ± 8

52 ± 7

 

333 µg

10 ± 1

13 ± 4

33 ± 5

136 ± 14

50 ± 8

 

1000 µg

10 ± 4

8 ± 3

33 ± 4

109 ± 4

47 ± 12

 

2500 µg

13 ± 3

8 ± 3

24 ± 4

57 ± 7

32 ± 5

 

5000 µg

13 ± 1* 

7 ± 3*

26 ± 7

40 ± 4

21 ± 2* 

2-AA

2.5 µg

372 ± 72

174 ± 11

3958 ± 855

4259 ± 350

 

2-AA

10 µg

 

 

 

 

298 ± 8

 * Reduced background count

Applicant's summary and conclusion

Conclusions:
The test substance is negative in Bacterial Reverse Mutation Assay for S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A with and without S9 metabolic activation, hence is classified as not mutagenic.
Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate. No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed reduced background growth in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used except strains TA 1537 and WP2 uvrA. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.