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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity and Cytotoxicity: Negative, OECD 471 (AMES test), Anon, 2016

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:
09-August-2016 to 31-August-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21-July-1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
31 March 2011
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Assumed stable. Stability at higher temperatures not availbale.
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

OTHER SPECIFICS: N/A
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO and saline solution
- Justification for choice of solvent/vehicle: Not reported
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Solution pre-incubated for 30 minutes prior to addition to 3 mL of molton top agar which was subsequently added to the selected agar plate.
- Cell density at seeding (if applicable): 10000000000 cells/mL

DURATION
- Preincubation period: 30 minutes
- Exposure duration: N/A
- Expression time (cells in growth medium): 48 ± 4 h
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: N/A

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: N/A
- Any supplementary information relevant to cytotoxicity: N/A

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER: N/A
Rationale for test conditions:
Preliminary test conducted at seven concentrations of the test item; 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the tester strains; TA1535, TA1537, TA98, TA100 and WP2uvrA.

Based on the results of the first pre-incubation assay, five doses (increasing with approximately half-log steps) of the test item were selected and tested in triplicate in each strain in the second pre-incubation assay.

The highest concentration of the test item used in the second mutation assay was the level at which the test item inhibited bacterial growth unless the test item.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.

b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
None.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
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:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
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:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
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:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
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:
negative
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
- Effects of pH: N/A
- Effects of osmolality: N/A
- Evaporation from medium: N/A
- Water solubility: N/A
- Precipitation: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in all tester strains.
- Definition of acceptable cells for analysis: No
- Other confounding effects: No

RANGE-FINDING/SCREENING STUDIES:

The test item was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: N/A

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: N/A
- Indication whether binucleate or mononucleate where appropriate: N/A

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: visual observation of bacteria lawn made using dissection microscope.
- Other observations when applicable: N/A

Table 1:       Experiment 1- Mutagenic response in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with
different strains ofSalmonella typhimuriumand oneEscherichia colistrain.


TA1535


TA1537

 

TA98

TA100

WP2uvrA

Without S-9 Mix

Positive control

967 ± 82

85 ± 79

1820 ± 203

783 ± 109

124 ± 33

Solvent control

14 ± 5

13 ± 79

17 ± 3

117 ± 18

20 ± 9

5.4

11 ± 2

15 ± 79

15 ± 6

139 ± 8

22 ± 5

17

11 ± 3n

8 ± 7n

17 ± 5n

114 ± 27n

18 ± 8n

52

0 ± 0a

0 ± 0a

0 ± 0a

0 ± 0a

15 ± 4m

164

0 ± 0a

0 ± 0a

0 ± 0a

0 ± 0a

0 ± 0a

512

0 ± 0a

0 ± 0a

0 ± 0a

0 ± 0a

0 ± 0a

1600

0 ± 0a NP

0 ± 0a NP

0 ± 0a NP

0 ± 0a NP

0 ± 0a NP

5000

0 ± 0a SP

0 ± 0a SP

0 ± 0a SP

0 ± 0a SP

0 ± 0a SP

With S-9 Mix

Positive control

144 ± 26

210 ± 12

693 ± 76

1337 ± 103

586 ± 44

Solvent control

14 ± 6

12 ± 1

23 ± 7

108 ± 9

31 ± 5

5.4

12 ± 10

19 ± 0

27 ± 5

131 ± 11

29 ± 8

17

12 ± 1n

11 ± 4n

27 ± 7n

116 ± 13n

37 ± 4

52

5 ± 3m

4 ± 3m

20 ± 2m

85 ± 3m

31 ± 3n

164

0 ± 0a

0 ± 0a

0 ± 0a

0 ± 0a

18 ± 2m

512

0 ± 0a

0 ± 0a

0 ± 0a

0 ± 0a

e MC a

1600

0 ± 0a NP

0 ± 0a NP

0 ± 0a NP

0 ± 0a NP

0 ± 0a NP

5000

0 ± 0a SP

0 ± 0a SP

0 ± 0a SP

0 ± 0a SP

0 ± 0a SP

1preincubation assay (5 % S9)

MCmicrocolonies

NPno precipitate

SPslight precipitate

abacterial background lawn absent

ebacterial background lawn extremely reduced

mbacterial background lawn moderately reduced

nnormal bacterial lawn

Table 2:       Experiment 1- Mutagenic response in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay (additional test)

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with
different strains ofSalmonella typhimuriumand oneEscherichia colistrain.


TA1535


TA1537

 

TA98

TA100

WP2uvrA

Without S-9 Mix

Positive control

109 6 ±79

97 ± 9

2088 ± 77

813 ± 23

231 ± 30

Solvent control

10 ± 4

2 ± 4

15 ± 4

107 ± 10

25 ± 4

0.056

17 ± 10

8 ± 3

15 ± 3

100 ± 17

21 ± 4

0.18

14 ± 2

3 ± 2

15 ± 2

98 ± 4

17 ± 6

0.55

16 ± 3

7 ± 5

19 ± 10

109 ± 4

23 ± 3

1.7

16 ± 9

6 ± 4

16 ± 5

102 ± 27

24 ± 3

5.4

12 ± 2n NP

3 ± 3n NP

14 ± 2n NP

97 ± 10n NP

25 ± 1n NP

With S-9 Mix

Positive control

127 ± 7

183 ± 17

737 ± 38

1696 ± 181

632 ± 33

Solvent control

14 ± 4

6 ± 1

22 ± 4

116 ± 26

35 ± 2

0.18

12 ± 4

4 ± 1

22 ± 2

103 ± 21

37 ± 8

0.55

15 ± 4

8 ± 5

20 ± 6

106 ± 1

34 ± 5

1.7

10 ± 2

12 ± 6

24 ± 5

105 ± 17

34 ± 5

5.4

16 ± 8n NP

7 ± 4n NP

24 ± 9n NP

107 ± 12n NP

38 ± 4n NP

1preincubation assay (5% S9)

NPno precipitate

nnormal bacterial background lawn

Table 3:       Experiment 2- Mutagenic response in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with
different strains ofSalmonella typhimuriumand oneEscherichia colistrain.


TA1535


TA1537

 

TA98

TA100

WP2uvrA

Without S-9 Mix

Positive control

837 ± 51

80 ± 15

1568 ± 44

663 ± 94

140 ± 45

Solvent control

7 ± 2

4 ± 1

13 ± 3

93 ± 7

19 ± 9

0.17

5 ± 2

4 ± 1

11 ± 4

94 ± 8

 

0.55

8 ± 3

2 ± 2

14 ± 4

92 ± 10

18 ± 3

1.7

5 ± 3

5 ± 5

14 ± 6

87 ± 4

22 ± 7

5.4

5 ± 3

4 ± 1

9 ± 3

90 ± 6

19 ± 4

17

6 ± 2n

4 ± 1n

12 ± 4n

89 ± 8n

23 ± 9n

52

e MC NP

e MC NP

0 ± 0a NP

e MC NP

11 ± 3m

164

-

-

-

-

0 ± 0a NP

With S-9 Mix

Positive control

71 ± 2

97 ± 8

335 ± 15

1432 ± 78

539 ± 33

Solvent control

7 ± 6

5 ± 2

19 ± 8

81 ± 16

25 ± 4

0.17

8 ± 3

5 ± 0

20 ± 3

82 ± 8

 

0.55

9 ± 4

5 ± 1

16 ± 4

86 ± 11

32 ± 8

1.7

4 ± 1

5 ± 3

20 ± 5

91 ± 16

35 ± 13

5.4

5 ± 1

5 ± 2

23 ± 4

70 ± 7

33 ± 8

17

5 ± 3n

8 ± 1

22 ± 2n

75 ± 13n

29 ± 1

52

e MC NP

0 ± 0a NP

e MC NP

e MC NP

30 ± 3n

164

-

-

-

-

e MC NP

1preincubation assay

MCmicrocolonies

NPno precipitate

abacterial background lawn absent

ebacterial background lawn extremely reduced

mbacterial background lawn moderately reduced

nnormal bacterial background lawn

- not tested

 

Conclusions:
Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The mutagenic and cytotoxic potential of the test substance was determined in accordance with OECD 471 guidance and GLP.

In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction in the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix.

In the first mutation test, not enough non-toxic dose levels were present in all five tester strains, therefore an additional experiment was performed to complete the data of the first mutation experiment. In this additional mutation experiment,the test item was tested at a concentration range of 0.056 to 5.4 µg/plate in the absence of S9-mix and at a range of 0.18 to 5.4 µg/plate in the presence of S9-mix. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation assay, the test item was tested up to concentrations of 52 and 164 µg/plate (absence and presence of 10% (v/v) S9-mix, respectively) in the tester strains TA1535, TA1537, TA100 and TA98, and up to 164 and 512 µg/plate (absence and presence of 10% (v/v) S9-mix, respectively) in tester strain WP2uvrA. The test item did not precipitate on the plates at these dose levels. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli 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

The mutagenic and cytotoxic potential of the test substance was determined in accordance with OECD 471 guidance and GLP.

In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction in the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix.

In the first mutation test, not enough non-toxic dose levels were present in all five tester strains, therefore anadditional experiment was performed to complete the data of the first mutation experiment. In this additional mutation experiment,the test itemwas testedat a concentration range of 0.056 to 5.4 µg/plate in the absence of S9-mix and at a range of 0.18 to 5.4 µg/plate in the presence of S9-mix. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation assay, the test item was tested up to concentrations of 52 and 164 µg/plate (absence and presence of 10% (v/v) S9-mix, respectively) in the tester strains TA1535, TA1537, TA100 and TA98, and up to 164 and 512 µg/plate (absence and presence of 10% (v/v) S9-mix, respectively) in tester strain WP2uvrA. The test item did not precipitate on the plates at these dose levels. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimuriumreverse mutation assay and in theEscherichia coli reverse mutation assay.

Justification for classification or non-classification

No classification criteria are met in accordance with Regulation (EC) No 1272/2008.