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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test

Under the conditions of this study, the test material was considered to be non-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:
23 March 2017 to 14 April 2017
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:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark over silica gel


Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS AND MEDIA USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
- Methods for maintenance in cell culture: All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS AND MEDIA USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
- Methods for maintenance in cell culture: All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Experiment 1, Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The maximum recommended dose level was used.
Experiment 2, Pre-Incubation Method: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The dose range used for Experiment 2 was determined by the results of Experiment 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene and 4-Nitroquinoline-1-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation

EXPERIMENT 1- PLATE INCORPORATION METHOD
- Dose Selection: Eight concentrations of the test material (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
- Without Metabolic Activation: 0.1 mL of the appropriate concentration of test material, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each condition was assayed using triplicate plates.
- With Metabolic Activation: The procedure was the same as described above except that following the addition of the test material formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

EXPERIMENT 2- PRE-INCUBATION METHOD
- Dose Selection: The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 5 to 5000 µg/plate. Seven test material dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test material following the change in test methodology from plate incorporation to pre-incubation.
- Without Metabolic Activation: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test material formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3°C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
- With Metabolic Activation: The procedure was the same as described above, except that following the addition of the test material and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3°C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

INCUBATION AND SCORING
For both experiments all of the plates were incubated at 37 ± 3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

ACCEPTABILITY CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are: TA1535: 7 to 40, TA100: 60 to 200, TA1537: 2 to 30, TA98: 8 to 60 and WP2uvrA: 10 to 60.
- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test material activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium, other: TA100, TA1535, TA98 and TA1537,
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRE- EXPERIMENT CHECKS
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test material formulation was also shown to be sterile.

VALIDITY OF THE CONTROLS
Results for the negative controls (concurrent untreated control plates performed on the same day as the Mutation Test) were considered to be acceptable.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

RESULTS
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 for Experiment 1 (plate incorporation) and Table 2 for Experiment 2 (pre-incubation).
The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method), the test material induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in the presence and absence of metabolic activation (S9-mix). Consequently the same maximum dose level was used in the second mutation test. The test material induced a slightly stronger toxic response in the second mutation test (employing the pre-incubation modification) with weakened bacterial background lawns noted from 500 µg/plate to all of the Salmonella strains dosed in the absence of S9-mix and at 5000 µg/plate to the Salmonella strains dosed in the presence of S9-mix and Escherichia coli strain WP2uvrA dosed in the presence and absence of S9-mix. A test material precipitate (particulate in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 150 µg/plate (TA1535 dosed in the absence of S9-mix) and to WP2uvrA in the second mutation test (absence of S9-mix) at the same dose level. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for each tester strain and the mean maximum fold increase was only 1.5 times the concurrent vehicle controls.

Table 1: Summary of Experiment 1

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

1.5

5

15

50

150

500

1500

5000

88

95

80

86

75

91

86

79

61

16

16

17

19

21

23*

17

20

13

29

30

30

31

26

22

21

21

17

32

30

26

26

24

19

20

20

11

13

13

13

18

16

13

10

11

4

+

Solvent

1.5

5

15

50

150

500

1500

5000

96

92

97

91

88

94

88

84

50

25

24

20

23

15

21

24

13

6

39

34

34

37

41

28

22

27

25

33

28

34

30

26

29

29

21

18

18

16

13

9

10

13

12

12

6

Positive Controls

-

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

Mean no. colonies/plate

665

417

790

240

255

+

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

Mean no. colonies/plate

514

234

329

98

313

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

* = P 0.05

Table 2: Summary of Experiment 2

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

5

15

50

150

500

1500

5000

87

95

91

95

85

81

86

61

14

17

11

13

20

20

14

9

23

32

29

23

34*

23

24

23

20

17

12

13

19

18

13

13

14

11

16

13

11

13

13

8

+

Solvent

5

15

50

150

500

1500

5000

110

107

111

109

107

102

92

68

16

16

12

14

16

13

10

8

31

31

33

32

39

35

31

19

21

16

20

16

21

18

24

13

10

12

11

13

10

11

13

8

Positive Controls

-

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

Mean no. colonies/plate

612

547

620

168

262

+

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

Mean no. colonies/plate

966

213

206

124

303

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

* = P 0.05

Conclusions:
Under the conditions of this study, the test material was considered to be non-mutagenic.
Executive summary:

The potential of the test material to cause cytotoxicity was determined in accordance with the standardised guidelines OECD 471, EU Method B13/14, USA EPA OCSPP870.5100 and The Japanese Regulatory Authorities including METI, MHLW and MAFF, under GLP conditions using the Bacterial Reverse Mutation Assay.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate. Seven test material dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test material following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method), the test material induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in the presence and absence of metabolic activation (S9-mix). Consequently the same maximum dose level was used in the second mutation test. The test material induced a slightly stronger toxic response in the second mutation test (employing the pre-incubation modification) with weakened bacterial background lawns noted from 500 µg/plate to all of the Salmonella strains dosed in the absence of S9-mix and at 5000 µg/plate to the Salmonella strains dosed in the presence of S9-mix and Escherichia coli strain WP2uvrA dosed in the presence and absence of S9-mix. A test material precipitate (particulate in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 150 µg/plate (TA1535 dosed in the absence of S9-mix) and to WP2uvrA in the second mutation test (absence of S9-mix) at the same dose level. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for each tester strain and the mean maximum fold increase was only 1.5 times the concurrent vehicle controls.

Under the conditions of this study, the test material was considered to be non-mutagenic.

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

Additional information

The potential of the test material to cause cytotoxicity was determined in accordance with the standardised guidelines OECD 471, EU Method B13/14, USA EPA OCSPP870.5100and The Japanese Regulatory Authorities including METI, MHLW and MAFF, under GLP conditions using the Bacterial Reverse Mutation Assay. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate. Seven test material dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test material following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method), the test material induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in the presence and absence of metabolic activation (S9-mix). Consequently the same maximum dose level was used in the second mutation test. The test material induced a slightly stronger toxic response in the second mutation test (employing the pre-incubation modification) with weakened bacterial background lawns noted from 500 µg/plate to all of the Salmonella strains dosed in the absence of S9-mix and at 5000 µg/plate to the Salmonella strains dosed in the presence of S9-mix and Escherichia coli strain WP2uvrA dosed in the presence and absence of S9-mix. A test material precipitate (particulate in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 150 µg/plate (TA1535 dosed in the absence of S9-mix) and to WP2uvrA in the second mutation test (absence of S9-mix) at the same dose level. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for each tester strain and the mean maximum fold increase was only 1.5 times the concurrent vehicle controls.

Under the conditions of this study, the test material was considered to be non-mutagenic.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.