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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2018

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
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
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: September 2015; signature: November 2015
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Experiment 1 (direct plate assay): 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Experiment 2 (pre-incubation assay): 52, 164, 512, 1600, 5000 μg/plate
Experiment 3 (pre-incubation assay): 52, 164, 512, 1600, 5000 μg/plate
Since the pre-incubation phase was not performed in closed vessels in experiment 2, a third experiment was perfomed within closed vessels.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item was soluble in dimethyl sulfoxide. Test item concentrations were used within 3 hours of preparation.
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: ICR-191 ; 2-aminoanthracene ; tert-butyl hydroperoxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation) ; Experiment 3 in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Experiment 1. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies were counted.
Experiment 2. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were
pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO. After the pre-incubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies were counted.
Experiment 3. The same conditions as Experiment 2 was utilised in Experiment 3, except for the usage of closed vessels for the exposure.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn) and reduction in the number of revertants
Rationale for test conditions:
Selection of an adequate range of doses was based on the first experiment (seven concentrations in triplicate) with all five tester strains, both with and without S9-mix considering cytotoxicity and precipitation. See tables for more information.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle 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 the tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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:
No formal hypothesis testing was done. See 'Any other information on materials and methods' and 'evaluation criteria' for details on the acceptability and evaluation criteria of the assay.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Test item was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
Test item was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1600 µg/plate and upwards and at the top dose of 5000 µg/plate at the end of the incubation period.
Experiment 2: Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 1600 µg/plate and upwards. Precipitation of the test item on the plates was observed at the end of the incubation period in the absence of S9-mix at concentrations of 1600 µg/plate and upwards and in the presence of S9-mix at the concentration of 5000 µg/plate.
Experiment 3: Precipitation of the test item on the plates was not observed at the start of the incubation period and at the end of the incubation period at the concentration of 5000 µg/plate.


RANGE-FINDING/SCREENING STUDIES:
Experiment 1 (direct plate assay) served as a range finding test in all species/strains for subsequent pre-incubation assays.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control values were within the laboratory historical control data ranges except the response for TA100 in the presence of S9-mix, Experiment 2. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (50 revertant colonies) when compared against relevant historical control data (54 revertant colonies), the validity of the test was considered to be not affected.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested

Table 1. Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay – Experiment 1

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.

 

TA1535

 

 

TA1537

 

 

TA98

 

 

TA100

 

 

WP2uvrA

 

 

Without S9-mix

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

903

± 84

 

687

± 21

 

1161

± 161

 

765

± 47

 

953

± 72

 

Solvent control

7

± 2

 

3

± 2

 

10

± 0

 

79

± 10

 

26

± 5

 

5.4

12

± 4

 

7

± 2

 

15

± 3

 

68

± 17

 

27

± 11

 

17

11

± 1

 

4

± 3

 

9

± 2

 

70

± 9

 

29

± 2

 

52

10

± 2

 

7

± 4

n

13

± 1

 

67

± 14

 

27

± 4

 

164

9

± 6

 

3

±3

 

15

± 5

 

55

± 13

 

34

± 14

 

512

6

± 4

 

3

± 4

n

8

± 5

 

8

± 5

 

27

± 3

 

1600

7

± 4

NP

3

± 2

NP

12

± 3

NP

30

± 4

NP

27

± 12

NP

5000

5

± 1

n SP

2

± 1

n SP

8

± 5

n SP

32

± 8

n SP

28

± 3

n SP

With S9-mix

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

903

± 84

687

± 21

1161

± 161

765

± 47

953

± 72

Solvent control

7

± 2

3

± 2

10

± 0

79

± 10

26

± 5

5.4

12

± 4

7

± 2

15

± 3

68

± 17

27

± 11

17

11

± 1

4

± 3

9

± 2

70

± 9

29

± 2

52

10

± 2

7

± 4

n

13

± 1

67

± 14

27

± 4

164

9

± 6

3

± 3

15

± 5

55

± 13

34

± 14

512

6

± 4

3

± 4

n

8

± 5

8

± 5

27

± 3

1600

7

± 4

NP

3

± 2

NP

12

± 3

NP

30

± 4

NP

27

± 12

NP

5000

5

± 1

n SP

2

± 1

n SP

8

± 5

n SP

32

± 8

n SP

28

± 3

n SP

NP No precipitate

SP Slight Precipitate

N Normal bacterial background lawn

 

Table 2. Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay – Experiment 2

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.

 

TA1535

 

 

TA1537

 

 

TA98

 

 

TA100

 

 

WP2uvrA

 

 

Without S9-mix

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

991

± 91

 

161

± 25

 

1432

± 326

 

439

± 38

 

186

± 10

 

Solvent control

12

± 5

 

5

± 4

 

22

± 8

 

63

± 5

 

22

± 4

 

52

7

± 3

n

10

± 0

n

17

± 5

n

57

± 7

 

29

± 8

 

164

5

± 5

s

7

± 4

s

13

± 5

s

55

± 6

n

19

± 2

 

512

10

± 0

s NP

5

± 4

m NP

18

± 4

s NP

27

± 8

s NP

26

± 9

NP

1600

5

s

s SP

2

± 2

m SP

18

± 3

s SP

31

± 3

s SP

24

± 6

SP

5000

7

± 4

s SP

3

± 1

m SP

12

± 3

s SP

27

± 14

s SP

21

± 6

n SP

With S9-mix

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

166

± 8

162

± 17

692

± 157

1520

± 82

496

± 10

Solvent control

7

± 3

4

± 1

28

± 4

50

± 3

32

± 11

52

11

± 5

7

± 2

29

± 7

56

± 6

32

± 10

164

9

± 9

8

± 2

n

26

± 6

102

± 62

NP

37

± 7

512

11

± 1

NP

4

± 1

s

25

± 9

42

± 6

m

42

± 3

1600

8

± 6

SP

0

± 1

m NP

16

± 2

NP

36

± 13

m NP

33

± 3

NP

5000

7

± 2

n SP

1

± 2

m SP

14

± 6

n SP

17

± 8

m SP

30

± 5

n SP

NP No precipitate

SP Slight Precipitate

m Bacterial background lawn moderately reduced

n Normal bacterial background lawn

s Bacterial background lawn slightly reduced

 

Table 3. Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay – Experiment 3

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.

 

TA1535

 

 

TA1537

 

 

TA98

 

 

TA100

 

 

WP2uvrA

 

 

Without S9-mix

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

873

± 72

 

200

± 23

 

974

± 80

 

876

± 44

 

176

± 32

 

Solvent control

9

± 2

 

4

± 2

 

12

± 2

 

121

± 6

 

21

± 2

 

52

16

± 8

 

7

± 3

9

± 4

 

101

± 2

 

19

± 4

 

164

10

± 2

 

6

± 1

 

11

± 1

 

104

± 15

 

41

± 30

 

512

14

± 2

 

6

± 3

14

± 5

 

89

± 11

 

26

± 15

 

1600

8

± 1

n NP

3

± 2

n NP

9

± 3

n SP

65

± 22

n NP

19

± 6

NP

5000

6

± 2

s SP i

9

± 7

s SP

20

± 4

s SP

82

± 4

s SP

19

± 1

n SP

With S9-mix

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

213

± 8

237

± 5

491

± 36

1689

± 121

564

± 15

Solvent control

12

± 2

11

± 1

22

± 2

117

± 17

27

± 3

52

23

± 19

8

± 3

21

± 11

105

± 9

29

± 7

164

12

± 3

5

± 2

n

18

± 6

93

± 10

n

48

± 31

512

14

± 9

NP

5

± 2

s

13

± 7

101

± 8

s

30

± 3

1600

9

± 5

SP

1

± 1

m NP

13

± 1

n NP

84

± 9

m NP

33

± 4

NP

5000

8

± 5

n SP

5

± 5

m SP

14

± 3

s SP

48

± 15

m SP

34

± 7

n SP

NP No precipitate

SP Slight Precipitate

i Plate infected

m Bacterial background lawn moderately reduced

n Normal bacterial background lawn

s Bacterial background lawn slightly reduced

Conclusions:
Interpretation of results:
negative

Under the conditions of this study, the test itme was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD TG 471, EU Method B.13/14 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP, to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat; incorporating the plate incorporation and pre-incubation methods) in both the presence and absence of S-9 metabolic activation system. The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). An additional experiment in closed vessels using pre-incubation using similar to the second experiment. In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strains TA1537 and TA100. In the second mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item was tested up to or beyond a precipitating dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in the absence of S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and in the presence of S9-mix in tester strains TA1537 and TA100. In the third mutation assay, the test item was tested within closed containers in the pre-incubation assay up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in the in tester strains TA1535, TA1537, TA98 and TA100 in the absence and presence of S9-mix. 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. The test item did not induce a toxicologically 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. These results were confirmed in two independently repeated experiments. Under the conditions of this study, it is concluded that that the test item was 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

OECD TG 471, 2018 - The study was performed to the requirements of OECD TG 471, EU Method B.13/14 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP, to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat; incorporating the plate incorporation and pre-incubation methods) in both the presence and absence of S-9 metabolic activation system. The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). An additional experiment in closed vessels using pre-incubation using similar to the second experiment. In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strains TA1537 and TA100. In the second mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item was tested up to or beyond a precipitating dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in the absence of S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and in the presence of S9-mix in tester strains TA1537 and TA100. In the third mutation assay, the test item was tested within closed containers in the pre-incubation assay up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in the in tester strains TA1535, TA1537, TA98 and TA100 in the absence and presence of S9-mix. 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. The test item did not induce a toxicologically 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. These results were confirmed in two independently repeated experiments. Under the conditions of this study, it is concluded that that the test item was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity