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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05.08.2016-26.08.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan
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
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II:
-- Strain TA 1535, TA 1537, TA 98, TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
-- Strain WP2 uvrA: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

- Justification: in the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. Since no relevant toxic effects were observed 5000 μg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades.
Vehicle / solvent:
- Vehicle: DMSO
- Justification: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in Experiment I and pre-incubation in Experiment II.

DURATION
- Exposure duration: 48h at 37°C

NUMBER OF REPLICATIONS: 3

ACCEPTANCE CRITERIA: The assay is considered acceptable if the following criteria are met:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above 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
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

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.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxic effects occurred with and without S9, no precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxic effects occurred with and without S9, no precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxic effects occurred without S9, no precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxic effects occurred with and without S9, no precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxic effects occurred with and without S9, no precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Detailed results

Experiment I

 

Without Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean±SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

12±4

12±5

30±5

170±11

40±5

Untreated

n.a.

12±4

11±5

31±2

201±15

42±11

Test item

3

11±3

11±2

26±6

195±21

41±9

 

10

11±5

10±3

33±4

203±1

50±5

 

33

10±5

12±5

28±2

195±7

46±6

 

100

12±3

12±4

34±13

194±8

45±8

 

333

9±1

8±2

30±5

190±14

47±9

 

1000

9±4

10±1

26±4

110±11

36±4

 

2500

11±2

9±4

20±2

67±9

42±8

 

5000

9±2R

6±1

22±5

60±4

42±8

NaN3

10

1299±36

 

 

 

4-NOPD

10

 

 

510±74

 

 

4-NOPD

50

 

74±3

 

 

 

MMS

2.0µL/plate

 

 

 

 

1045±58

 

With Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean +/- SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

12±1

12±2

43±9

196±8

55±11

Untreated

n.a.

12±4

16±1

42±12

206±11

53±5

Test item

3

12±1

14±4

36±5

149±9

49±6

 

10

13±4

13±1

40±5

153±10

47±8

 

33

13±1

13±6

38±2

161±9

48±8

 

100

 16±2

15±5

36±9

168±5

62±6

 

333

11±5

17±7

42±8

156±24

54±6

 

1000

12±2

12±6

41±13

143±8

56±5

 

2500

12±4

15±4

46±7

129±8

57±8

 

5000

15±1

11±3

38±8

146±10

54±3

2-AA

2.5

423±23

352±35

4519±230

5580±151

 

2-AA

10

 

 

 

 

394±32

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

R: Reduced background growth

Experiment II

 

Without Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean±SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

10±4

10±4

24±6

142±5

32±5

Untreated

n.a.

7±2

9±2

28±8

198±6

41±8

Test item

3

 9±1

8±1

24±7

111±6

 41±3

 

10

 12±5

10±2

27±4

131±8

 35±5

 

33

10± 5

10± 1

20± 7

126± 8

 38± 8

 

100

9±2

10±3

20±7

128±2

43±9

 

333

10±2

7±3

19±5

52±8

28±7

 

1000

12±4

11±3

14±4

48±5

33±1

 

2500

8±2

7±1

5±1

7±2

15±5

 

5000

0±1

2±2

0±1

5±1

3±1MR

NaN3

10

1113±42

1891±172

4-NOPD

10

 

416±52

 

 

4-NOPD

50

 

83±16

 

 

 

MMS

2.0µL/plate

 

 

 

 

724±12

 

With Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean +/- SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

8±3

12±2

28±2

139±6

46±10

Untreated

n.a.

11±4

15±6

33±3

188±2

57±6

 Test item

3

 9±3

14±2

36±5

111±26

 41±3

 

10

7±2

16±6

28±8

127±9

 50±11

 

33

8±3

10±3

35±4

125±18

49±9

 

100

10±1

13±3

37±5

126±1

44±3

 

333

9±2

13±5

30±5

97±11

51±10

 

1000

10±5

9±5

27±3

120±16

40±11

 

2500

8±3
12±4

27±2

61±5

39±7

 

5000

8±3MR

 6±2MR

 20±4

 34±10R

37±7R

2-AA

2.5

335±9

214±11

2619±25

3712±269

 

2-AA

10

 

 

 

 

363±27

 

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

R: Reduced background growth

M Manual count

Conclusions:
The test item is not mutagenic.
Executive summary:

In the current study the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) was assessed according to OECD 471 and GLP.

The Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA were used.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate.

The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 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, except for strain TA98.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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, 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 and is considered to be non-mutagenic in the reverse mutation assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For this endpoint there is 1 study available in which the potential of the test item to induce gene mutations was assessed according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) according to OECD 471 and GLP. TheSalmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA were used with and without liver microsomal activation. The assay was performed in two independent experiments and each concentration, including the controls, was tested in triplicate. In the Experiment I and II the test item was tested at 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate.

No precipitation of the test item occurred up to the highest investigated dose.


Toxic effects, evident as a reduction in the number of revertants, occurred in all strains. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

The test item did not induce gene mutations and is considered to be non-mutagenic in the reverse mutation assay.

Justification for classification or non-classification

The test item did not induce gene mutations in a reverse mutation assay Ames test and is therefore considered to be non-mutagenic.