Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the Ames test, the substance is 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:
From June 14, 2017 to July 11, 2017
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)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate
The maximum concentration was 5000 µg/plate (the maximum recommended dose level)
Vehicle / solvent:
The test substance was fully miscible in sterile distilled water at 50 mg/mL in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle.
Untreated negative controls:
yes
Remarks:
Identity: Sterile distilled water, Supplier: Aguettant, Batch number (purity): 3012436 (N/A), Expiry: 10/2018
Negative solvent / vehicle controls:
yes
Remarks:
Identity: Sterile distilled water, Supplier: Aguettant, Batch number (purity): 3012436 (N/A), Expiry: 10/2018
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: Identity: 2-Aminoanthracene (2AA), CAS No.: 613-13-8
Details on test system and experimental conditions:
Test System and Supporting Information
Bacteria: The five strains of bacteria used, and their mutations, are as follows:
Salmonella typhimurium
Strains: Genotype: Type of mutations indicated
TA1537: his C 3076; rfa-; uvrB-: frame shift
TA98: his D 3052; rfa-; uvrB-;R-factor: frame shift
TA1535: his G 46; rfa-; uvrB-: base-pair substitution
TA100: his G 46; rfa-; uvrB-;R-factor: base-pair substitution

Escherichia coli
Strain: Genotype: Type of mutations indicated
WP2uvrA: trp-; uvrA-: base-pair substitution
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000). The bacteria used in the test were obtained from: 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. 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.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 6147883 03/21) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 44673 07/17 and 44674 07/17).

Study controls
The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate. The positive control substances used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix;
and The maximum dosing solution of the test substance in the absence of S9-mix only (test in singular only).
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:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. 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 substance 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 substance 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:
other: S. typhimurium TA 1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Results

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 substance formulation was also shown to be sterile. These data are not given in the report. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data were for concurrent untreated control plates performed on the same day as the Mutation Test. The vehicle (sterile distilled water) 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 substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method).

Table1: Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 (Plate Incorporation)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

100

 

24

 

25

 

18

 

8

 

93

(91)

20

(20)

18

(24)

18

(18)

15

(12)

80

 

16

 

28

 

18

 

14

 

Experiment 2 (Pre-Incubation)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

91

 

21

 

34

 

26

 

15

 

96

(90)

12

(16)

29

(29)

19

(21)

10

(12)

83

 

16

 

25

 

19

 

12

 

Table 2 Test Results: Experiment 1 – Without Metabolic Activation(Plate Incorporation)

Test Period

From: 26 June 2017

To: 29 June 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Water)

114

93

118

(108)

13.4#

21

21

23

(22)

1.2

22

28

20

(23)

4.2

17

20

20

(19)

1.7

15

13

12

(13)

1.5

1.5 µg

104

94

103

(100)

5.5

16

22

15

(18)

3.8

24

30

20

(25)

5.0

22

22

18

(21)

2.3

12

7

12

(10)

2.9

5 µg

110

131

119

(120)

10.5

28

25

28

(27)

1.7

18

24

26

(23)

4.2

24

18

20

(21)

3.1

18

24

12

(18)

6.0

15 µg

104

99

124

(109)

13.2

22

24

14

(20)

5.3

21

19

29

(23)

5.3

20

17

26

(21)

4.6

9

15

9

(11)

3.5

50 µg

76

97

98

(90)

12.4

19

21

21

(20)

1.2

24

14

31

(23)

8.5

24

23

27

(25)

2.1

9

13

10

(11)

2.1

150 µg

99

97

99

(98)

1.2

19

24

16

(20)

4.0

26

18

24

(23)

4.2

18

14

20

(17)

3.1

9

15

9

(11)

3.5

500 µg

102

107

99

(103)

4.0

24

25

14

(21)

6.1

27

22

26

(25)

2.6

15

25

19

(20)

5.0

10

14

13

(12)

2.1

1500 µg

78

82

100

(87)

11.7

27

18

25

(23)

4.7

25

31

28

(28)

3.0

23

20

25

(23)

2.5

17

11

13

(14)

3.1

5000 µg

102

104

102

(103)

1.2

25

23

16

(21)

4.7

26

19

24

(23)

3.6

18

25

18

(20)

4.0

10

16

13

(13)

3.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

501

541

622

(555)

61.6

502

478

470

(483)

16.7

281

269

308

(286)

20.0

177

196

188

(187)

9.5

177

329

245

(250)

76.1

Table 3 Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period

From: 26 June 2017

To: 29 June 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Water)

113

103

106

(107)

5.1#

22

20

22

(21)

1.2

18

26

24

(23)

4.2

27

20

25

(24)

3.6

11

10

12

(11)

1.0

1.5 µg

92

103

113

(103)

10.5

21

11

19

(17)

5.3

17

20

22

(20)

2.5

27

21

26

(25)

3.2

8

8

10

(9)

1.2

5 µg

93

92

95

(93)

1.5

31

13

22

(22)

9.0

16

22

20

(19)

3.1

32

26

31

(30)

3.2

8

10

11

(10)

1.5

15 µg

110

93

92

(98)

10.1

20

19

17

(19)

1.5

23

24

16

(21)

4.4

22

22

22

(22)

0.0

9

7

8

(8)

1.0

50 µg

93

91

95

(93)

2.0

22

20

17

(20)

2.5

23

14

22

(20)

4.9

16

24

23

(21)

4.4

6

8

9

(8)

1.5

150 µg

91

111

87

(96)

12.9

16

23

23

(21)

4.0

19

20

20

(20)

0.6

20

22

20

(21)

1.2

14

4

10

(9)

5.0

500 µg

109

89

100

(99)

10.0

20

21

21

(21)

0.6

17

27

16

(20)

6.1

32

32

13

(26)

11.0

11

10

14

(12)

2.1

1500 µg

90

102

92

(95)

6.4

21

22

22

(22)

0.6

19

20

24

(21)

2.6

29

23

30

(27)

3.8

9

11

10

(10)

1.0

5000 µg

85

100

82

(89)

9.6

24

15

21

(20)

4.6

21

18

18

(19)

1.7

24

25

30

(26)

3.2

11

11

11

(11)

0.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

850

818

865

(844)

24.0

189

224

245

(219)

28.3

393

496

412

(434)

54.8

198

213

231

(214)

16.5

400

412

423

(412)

11.5

Table 4 Test Results: Experiment 2 – Without Metabolic Activation(Pre-Incubation)

Test Period

From: 07 July 2017

To: 10 July 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Water)

103

94

115

(104)

10.5#

17

10

17

(15)

4.0

27

25

29

(27)

2.0

24

21

32

(26)

5.7

17

11

10

(13)

3.8

15 µg

76

103

82

(87)

14.2

11

13

11

(12)

1.2

28

25

22

(25)

3.0

32

36

19

(29)

8.9

4

13

16

(11)

6.2

50 µg

89

76

122

(96)

23.7

12

11

17

(13)

3.2

19

20

28

(22)

4.9

32

17

25

(25)

7.5

7

13

13

(11)

3.5

150 µg

109

114

94

(106)

10.4

12

12

16

(13)

2.3

35

28

28

(30)

4.0

24

32

24

(27)

4.6

8

11

15

(11)

3.5

500 µg

112

102

108

(107)

5.0

17

16

17

(17)

0.6

20

22

37

(26)

9.3

25

21

21

(22)

2.3

15

17

18

(17)

1.5

1500 µg

119

103

118

(113)

9.0

18

14

14

(15)

2.3

34

22

31

(29)

6.2

17

26

25

(23)

4.9

12

10

11

(11)

1.0

5000 µg

90

98

85

(91)

6.6

8

8

10

(9)

1.2

31

26

24

(27)

3.6

22

22

22

(22)

0.0

11

14

12

(12)

1.5

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

824

885

772

(827)

56.6

594

576

778

(649)

111.8

767

721

759

(749)

24.6

394

439

469

(434)

37.7

461

365

598

(475)

117.1

Table 5 Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period

From: 07 July 2017

To: 10 July 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Water)

90

107

92

(96)

9.3#

11

14

13

(13)

1.5

40

36

27

(34)

6.7

22

35

22

(26)

7.5

17

12

13

(14)

2.6

15 µg

88

110

106

(101)

11.7

11

11

9

(10)

1.2

24

29

30

(28)

3.2

23

24

22

(23)

1.0

17

10

18

(15)

4.4

50 µg

100

104

120

(108)

10.6

11

11

11

(11)

0.0

21

39

37

(32)

9.9

30

15

19

(21)

7.8

10

8

10

(9)

1.2

150 µg

106

131

92

(110)

19.8

12

7

11

(10)

2.6

26

22

33

(27)

5.6

30

26

27

(28)

2.1

9

18

9

(12)

5.2

500 µg

102

100

100

(101)

1.2

8

11

11

(10)

1.7

32

31

31

(31)

0.6

24

23

27

(25)

2.1

10

11

12

(11)

1.0

1500 µg

97

94

122

(104)

15.4

8

15

7

(10)

4.4

31

36

27

(31)

4.5

27

28

31

(29)

2.1

9

12

15

(12)

3.0

5000 µg

86

84

86

(85)

1.2

11

6

11

(9)

2.9

28

29

32

(30)

2.1

33

27

35

(32)

4.2

15

14

13

(14)

1.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1182

1403

1213

(1266)

119.7

301

258

232

(264)

34.8

193

157

187

(179)

19.3

191

182

169

(181)

11.1

368

361

391

(373)

15.7

Conclusions:
Under the conditions of the Ames test, the substance was determined to be non-mutagenic with or without metabolic activation.
Executive summary:

A study was conducted to determine the genotoxic potential of the test substance (purity: assumed to be >96%) in Salmonella typhimurium and Escherichia coli according to OECD Guideline 471, EU Method B13/14 and the USA, EPA OCSPP Harmonized Guideline, Bacterial Reverse Mutation Test (Ames Test), in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the substance using both the Ames plate incorporation and the pre-incubation methods at up to eight dose levels (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 ranged from 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended to 15 to 5000 µg/plate, following the results of Experiment 1. Six test substance concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test substance following the change in test methodology. The vehicle (sterile distilled water) 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 substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method. Under the study conditions, the test substance was determined to be non-mutagenic in the Ames test, with or without metabolic activation (Envigo, 2017).

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

Additional information

A study was conducted to determine the genotoxic potential of the test substance (purity: assumed to be >96%) in Salmonella typhimurium and Escherichia coli according to OECD Guideline 471, EU Method B13/14 and the USA, EPA OCSPP Harmonized Guideline, Bacterial Reverse Mutation Test (Ames Test), in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the substance using both the Ames plate incorporation and the pre-incubation methods at up to eight dose levels (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 ranged from 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended to 15 to 5000 µg/plate, following the results of Experiment 1. Six test substance concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test substance following the change in test methodology. The vehicle (sterile distilled water) 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 substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method. Under the study conditions, the test substance was determined to be non-mutagenic in the Ames test, with or without metabolic activation (Envigo, 2017).

Justification for classification or non-classification

Based on negative results in an in vitro Ames test, the substance does not warrant classification for genotoxicity according to the EU CLP criteria (Regulation 1272/2008/EC).​​