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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

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:
08 - 27 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 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
Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
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: 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 the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA), methylmethanesulfonate (MMS)
Remarks:
+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: sodium azide (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)
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 substance is considered as 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 deviation were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp. 2: -S9 and +S9: starting at 2500 µg/plate
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:
exp. 1: +S9: at 5000 µg/plate; exp. 2: -S9 and +S9: starting at 2500 µg/plate
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:
exp. 2: -S9 and +S9: starting at 2500 µg/plate
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:
exp. 1: -S9 and +S9: at 5000 µg/plate; exp. 2: -S9 and +S9: starting at 2500 µg/plate
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:
exp. 2: -S9 and +S9: starting at 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the overlay agar in the test tubes at 5000 µg/plate with S9 mix. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed at 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Reduced background growth was observed at the higher concentrations with and without metabolic activation in Experiment II. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in both experiments. Please refer to table 3 and 4.

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)

18 ± 3

150 ± 17

41 ± 6

15 ± 2

22 ± 1

-

0

13 ± 4

150 ± 16

37 ± 7

15 ± 3

30 ± 2

-

3

16 ± 2

150 ± 18

42 ± 5

16 ± 1

29 ± 6

-

10

14 ± 5

139 ± 13

44 ± 7

16 ± 6

27 ± 6

-

33

13 ± 2

116 ± 32

50 ± 11

12 ± 4

30 ± 5

-

100

17 ± 4

155 ± 12

38 ± 3

18 ± 2

28 ± 6

-

333

20 ± 4

133 ± 27

39 ± 4

10 ± 3

26 ± 1

-

1000

21 ± 2

129 ± 21

31 ± 5

13 ± 4

25 ± 4

-

2500

17 ± 5

82 ± 5

23 ± 7

15 ± 2

30 ± 3

-

5000

10 ± 3

64 ± 9

25 ± 5

16 ± 5

18 ± 3

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

(μg/plate)

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

957 ± 12

2018 ± 42

819 ± 36

100 ± 18

409 ± 24

+

0 (DMSO)

13 ± 3

138 ± 10

49 ± 2

16 ± 5

30 ± 4

+

0

16 ± 4

160 ± 16

63 ± 17

18 ± 2

33 ± 12

+

3

13 ± 7

121 ± 16

58 ± 15

14 ± 1

32 ± 10

+

10

12 ± 5

140 ± 5

49 ± 7

13 ± 4

35 ± 3

+

33

16 ± 6

135 ± 20

45 ± 8

14 ± 5

42 ± 7

+

100

16 ± 4

142 ± 16

58 ± 8

12 ± 3

40 ± 5

+

333

14 ± 6

125 ± 21

62 ± 8

16 ± 4

41 ± 8

+

1000

15 ± 3

123 ± 6

37 ± 5

13 ± 4

47 ± 10

+

2500

19 ± 7

63 ± 7

28 ± 8

15 ± 4

44 ± 7

+

5000

19 ± 2P

38 ± 0P

26 ± 7P

3 ± 1P M

30 ± 3P

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

405 ± 49

3237 ± 49

457 ± 4

144 ± 9

4429 ± 353

NaN3: Sodium azide

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

MMS: Methylmethanesulfonate

2-AA: 2-aminoanthracene

M: Manual count

P: Precipitate

 

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

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)

10 ± 3

146 ± 11

27 ± 6

10 ± 1

21 ± 5

-

0

10 ± 2

188 ± 5

40 ± 5

7 ± 2

22 ± 1

-

10

10 ± 4

154 ± 7

41 ± 7

11 ± 2

22 ± 4

-

33

12 ± 2

150 ± 14

42 ± 2

13 ± 5

25 ± 7

-

100

14 ± 2

145 ± 18

45 ± 11

12 ± 4

24 ± 2

-

333

12 ± 0

137 ± 17

34 ± 1

13 ± 3

29 ± 1

-

1000

15 ± 5

95 ± 15

41 ± 3

13 ± 0

25 ± 4

-

2500

11 ± 4M R

56 ± 17M R

12 ± 5M R

8 ± 4M R

11 ± 2M R

-

5000

6 ± 2M R

16 ± 14M R

3 ± 2M R

6 ± 2M R

0 ± 0M R

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

(μg/plate)

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

977 ± 166

1851 ± 209

475 ± 29

103 ± 3

432 ± 12

+

0 (DMSO)

13 ± 2

146 ± 7

47 ± 8

13 ± 3

33 ± 7

+

0

12 ± 2

139 ± 33

57 ± 7

13 ± 4

31 ± 8

+

10

12 ± 3

133 ± 8

54 ± 3

14 ± 4

31 ± 8

+

33

12 ± 5

131 ± 16

42 ± 7

11 ± 2

38 ± 6

+

100

13 ± 5

142 ± 16

48 ± 4

9 ± 1

32 ± 3

+

333

11 ± 2

111 ± 6

50 ± 14

9 ± 2

29 ± 2

+

1000

12 ± 4

88 ± 17

54 ± 11

12 ± 3

35 ± 6

+

2500

4 ± 2M R

25 ± 7M R

27 ± 2M R

0 ± 0M R

14 ± 2M R

+

5000

0 ± 0M P R

0 ± 0M P R

18 ± 4M P R

0 ± 0M P R

14 ± 3M P R

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

421 ± 22

4628 ± 190

429 ± 14

181 ± 27

5196 ± 206

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 3. Reduced background growth in Experiment I and II at different concentrations (µg/plate).

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

/

/

2500 - 5000

2500 - 5000

TA 1537

/

/

2500 - 5000

2500 - 5000

TA 98

/

/

2500 - 5000

2500 - 5000

TA 100

/

/

2500 - 5000

2500 - 5000

WP2 uvrA

/

/

2500 - 5000

2500 - 5000

/ = no reduced background

Table 4. Reduction in the number of spontaneous revertants (below the induction factor of 0.5) in Experiment I and II at different concentrations (µg/plate).

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

/

/

/

2500 - 5000

TA 1537

/

5000

/

2500 - 5000

TA 98

/

/

5000

2500 - 5000

TA 100

5000

5000

2500 - 5000

2500 - 5000

WP2 uvrA

/

/

5000

5000

/ = no toxic effects

In experiment II, the number of colonies did not quite reach the lower limit of our historical control data in the negative control of strain WP2 uvrA without metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Conclusions:
Under the conditions of the Ames 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 test item precipitated in the overlay agar in the test tubes at 5000μg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000μg/plate with S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed reduced background growth in all strains used with and without S9 mix. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2015). In this study 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.

Justification for classification or non-classification

Based on the available data, there is no indication that the substance induces genetic toxicity in bacteria. The available data on genotoxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008