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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:
19 Aug - 23 Sep 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)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.

The pre-experiment is reported as Experiment I.

Experiment II: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to bacteria.
Controls
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
A test item is considered as a mutagenic 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.
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in strains TA 1537, TA 98 and TA 100 with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
but tested up to the precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate with and without S9 mix and in Experiment II from 2500 to 5000 µg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in Experiment I at 5000 µg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed reduced background growth in strains TA 1537, TA 98 and TA 100 with and without metabolic activation. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 1537 with metabolic activation (experiment 2) and in strains TA 98 and TA 100 with and without metabolic activation (experiment 2 and experiment 1 and 2. respectively). Please refer to table 3 and 4.

Any other information on results incl. tables

Table 1. Test results of main test 1 (plate incorporation).

With or without S9-Mix

Test substance concentration [μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

11 ± 3

154 ± 16

35 ± 1

9 ± 1

26 ± 6

-

0

13 ± 3

172 ± 9

38 ± 5

14 ± 0

23 ± 4

-

3

12 ± 2

160 ± 23

38 ± 6

9 ± 2

26 ± 4

-

10

12 ± 3

167 ± 16

43 ± 3

8 ± 3

25 ± 2

-

33

10 ± 2

161 ± 8

41 ± 10

8 ± 2

25 ± 3

-

100

9 ± 1

138 ± 24

38 ± 3

9 ± 1

21 ± 7

-

333

9 ± 1

56 ± 7R

39 ± 10

9 ± 3R

31 ± 2R

-

1000

8 ± 2

55 ± 4R

37 ± 9

6 ± 2R

27 ± 4R

-

2500

7 ± 2

54 ± 12R

37 ± 6

5 ± 2R

25 ± 5R

-

5000

10 ± 4P

63 ± 8P,R

33 ± 4P

5 ± 2P,R

20 ± 5P,R

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

1136 ± 68

2125 ± 126

829 ± 40

78 ± 6

336 ± 30

+

0 (DMSO)

11 ± 3

141 ± 7

48 ± 5

14 ± 2

30 ± 3

+

0

11 ± 4

170 ± 2

56 ± 11

13 ± 1

27 ± 4

+

3

11 ± 1

142 ± 11

49 ± 7

13 ± 2

30 ± 3

+

10

11 ± 4

171 ± 19

47 ± 5

11 ± 3

32 ± 2

+

33

10 ± 5

139 ± 14

43 ± 3

11 ± 2

37 ± 9

+

100

9 ± 3

150 ± 17

50 ± 5

13 ± 2

36 ± 2

+

333

7 ± 1

119 ± 24

46 ± 8

15 ± 1

32 ± 3

+

1000

9 ± 3

38 ± 3R

43 ± 8

12 ± 1M,R

29 ± 6R

+

2500

10 ± 4

33 ± 2M,R

46 ± 2

13 ± 1M,R

23 ± 2R

+

5000

12 ± 2P

29 ± 2P,M,R

37 ± 6P

13 ± 2P,M,R

16 ± 2P,M,R

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

348 ± 19

3985 ± 50

384 ± 50

197 ± 5

4320 ± 413

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

P: precipitate

R: reduced background growth

 

Table 2. Test results of main test 2 (preincubation).

With or without S9-Mix

Test substance concentration

g/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

12 ± 3

143 ± 9

36 ± 6

8 ± 2

30 ± 10

-

0

10 ± 3

201 ± 2

39 ± 3

7 ± 3

26 ± 9

-

3

10 ± 5

141 ± 3

43 ± 4

7 ± 1

22 ± 3

-

10

11 ± 5

132 ± 21

47 ± 4

7 ± 2

26 ± 2

-

33

9 ± 1

152 ± 10

39 ± 7

9 ± 2

28 ± 3

-

100

10 ± 3

54 ± 10

38 ± 6

7 ± 2

22 ± 2

-

333

10 ± 3

58 ± 10

24 ± 2

9 ± 2

16 ± 1

-

1000

9 ± 1

56 ± 8

41 ± 1

8 ± 1

7 ± 3M,R

-

2500

13 ± 4

55 ± 3

28 ± 10

10 ± 2

6 ± 2M,R

-

5000

7 ± 3

38 ± 11M,R

37 ± 10

6 ± 0R

5 ± 2M,R

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

905 ± 35

2051 ± 72

671 ± 107

79 ± 8

374 ± 50

+

0 (DMSO)

12 ± 2

115 ± 8

47 ± 9

9 ± 3

36 ± 2

+

0

9 ± 4

190 ± 29

59 ± 11

11 ± 3

32 ± 7

+

3

12 ± 2

126 ± 34

51 ± 5

12 ± 2

34 ± 11

+

10

8 ± 2

119 ± 21

53 ± 6

13 ± 1

37 ± 11

+

33

13 ± 1

128 ± 11

52 ± 10

11 ± 2

36 ± 7

+

100

13 ± 2

110 ± 18

54 ± 8

9 ± 5

43 ± 9

+

333

13 ± 3

64 ± 18

56 ± 17

13 ± 2

40 ± 11

+

1000

7 ± 3

42 ± 6

57 ± 16

13 ± 1

33 ± 8

+

2500

8 ± 2

22 ± 9M,R

43 ± 4

8 ± 2M,R

13 ± 2M,R

+

5000

13 ± 1

2 ± 2M,R

45 ± 4

4 ± 2M,R

5 ± 1M,R

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

367 ± 24

3181 ± 117

421 ± 12

185 ± 12

3909 ± 258

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

R: reduced background growth

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.
Executive summary:

The mutagenicity of the test item was studied with five mutant strains of Salmonella typhimurium

(TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) with and without metabolic activation according to OECD 471 under GLP conditions. Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.