Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the in vitro Ames test, the test substance, 'iso and anteiso C10-40 AAP EDM-ES', was considered to be non mutagenic 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:
From September 20, 2017 to November 05, 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:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
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
Additional strain / cell type characteristics:
not specified
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 (Presence and absence of S9)
Experiment 2: TA100 and TA1535 (presence and absence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate; TA98 and TA1537 (presence and absence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate; WP2uvrA (presence and absence of S9): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
The maximum concentration was 5000 µg/plate (the maximum recommended dose level).
Vehicle / solvent:
In solubility checks performed in–house, the test item was noted as insoluble sterile distilled water at 50 mg/mL but fully soluble in dimethyl sulphoxide at the same concentration. Dimethyl sulphoxide was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Identity: Dimethyl sulphoxide, Supplier: Fisher Scientific, Batch number (purity): 1710280 (>99%), Expiry: 06/2022
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, Batch number: STBB1901M9, Purity: 97.5%, Expiry date: 08 October 2019, Solvent: DMSO, Concentration: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 , 10 µg/plate for WP2uvrA
Details on test system and experimental conditions:
Bacteria
The five strains of bacteria used, and their mutations, are as follows:
1) Salmonella typhimurium
Strains - Genotype - Type of mutations indicated
TA1537 - his C 3076; rfa-; uvrB-: - frame shift
TA98 - his D 3052; rfa-; uvrB-; - R-factor
TA1535 - his G 46; rfa-; uvrB-: - base-pair substitution
TA100 - his G 46; rfa-; uvrB-;R-factor

2) 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:
1) University of California, Berkeley, on culture discs, on 04 August 1995.
2) 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 h. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
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 h. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
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. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are 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 individual plate counts, the mean number of revertant colonies and the standard deviations, for the test substance, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 and Table 2 for Experiment 1 (plate incorporation) and Table 3 and Table 4 for Experiment 2 (pre-incubation).

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

Test Period

From: 09 October 2017

16 October 2017†

To: 12 October 2017

19 October 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

(DMSO)

109

102

64

(92)

24.2#

33

35

31

(33)

2.0

19

29

32

(27)

6.8

24

28

22

(25)

3.1

14

18

23

(18)

4.5

1.5 µg

95

81

96

(91)

8.4

28

36

31

(32)

4.0

26

16

19

(20)

5.1

18

35

26

(26)

8.5

12

19

16

(16)

3.5

5 µg

91

88

95

(91)

3.5

31

29

27

(29)

2.0

25

19

21

(22)

3.1

18

42

11

(24)

16.3

14

12

13

(13)

1.0

15 µg

98

104

94

(99)

5.0

33

38

25

(32)

6.6

22

13

16

(17)

4.6

25

16

25

(22)

5.2

6

7

17

(10)

6.1

50 µg

79

87

79

(82)

4.6

28

29

30

(29)

1.0

29

25

23

(26)

3.1

25

23

20

(23)

2.5

18

16

11

(15)

3.6

150 µg

15 S

23 S

22 S

(20)

4.4

21 S

20 S

26 S

(22)

3.2

27

16

17

(20)

6.1

14

19

13

(15)

3.2

14

13

18

(15)

2.6

500 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

16

14

21

(17)

3.6

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

1500 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

13 S

15 S

23 S

(17)

5.3

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

5000 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

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

718

691

681

(697)

19.1

1381

1521

1499

(1467)

75.3

1043

938

1028

(1003)

56.8

189

218

180

(196)

19.9

231

253

227

(237)

14.0

Table 2: Test Results: Experiment 1 – With Metabolic Activation(Plate Incorporation)

Test Period

From: 09 October 2017

16 October 2017†

To: 12 October 2017

19 October 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

(DMSO)

84

77

70

(77)

7.0#

36

38

35

(36)

1.5

35

32

22

(30)

6.8

29

22

29

(27)

4.0

12

7

12

(10)

2.9

1.5 µg

78

61

68

(69)

8.5

40

37

37

(38)

1.7

31

35

32

(33)

2.1

25

24

35

(28)

6.1

15

9

12

(12)

3.0

5 µg

67

77

73

(72)

5.0

38

34

39

(37)

2.6

32

27

27

(29)

2.9

27

26

26

(26)

0.6

15

10

15

(13)

2.9

15 µg

70

80

79

(76)

5.5

40

40

40

(40)

0.0

27

31

25

(28)

3.1

21

27

26

(25)

3.2

11

16

16

(14)

2.9

50 µg

62

50

65

(59)

7.9

36

35

37

(36)

1.0

25

28

31

(28)

3.0

24

27

23

(25)

2.1

18

21

6

(15)

7.9

150 µg

42 S

57 S

45 S

(48)

7.9

33 S

32 S

38 S

(34)

3.2

19

21

25

(22)

3.1

21

19

21

(20)

1.2

9

18

13

(13)

4.5

500 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

21

27

21

(23)

3.5

7 S

11 S

10 S

(9)

2.1

7 S

12 S

9 S

(9)

2.5

1500 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

11 S

16 S

16 S

(14)

2.9

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

5000 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

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

2309

2082

2182

(2191)

113.8

422

349

390

(387)

36.6

467

376

480

(441)

56.7

270

267

270

(269)

1.7

502

417

434

(451)

45.0

Table 3: Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period

From: 02 November 2017

To: 05 November 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

(DMSO)

98

107

85

(97)

11.1#

11

14

13

(13)

1.5

20

21

20

(20)

0.6

18

26

25

(23)

4.4

10

12

14

(12)

2.0

0.15 µg

97

94

102

(98)

4.0

8

11

15

(11)

3.5

N/T

N/T

N/T

0.5 µg

100

92

79

(90)

10.6

16

13

18

(16)

2.5

N/T

29

16

21

(22)

6.6

18

14

8

(13)

5.0

1.5 µg

88

98

98

(95)

5.8

9

13

13

(12)

2.3

29

25

21

(25)

4.0

23

18

30

(24)

6.0

13

14

17

(15)

2.1

5 µg

95

93

74

(87)

11.6

11

10

10

(10)

0.6

30

17

24

(24)

6.5

18

34

19

(24)

9.0

9

8

5

(7)

2.1

15 µg

67 S

70 S

62 S

(66)

4.0

12

10

10

(11)

1.2

26

22

20

(23)

3.1

20

20

21

(20)

0.6

13

10

9

(11)

2.1

50 µg

62 S

58 S

63 S

(61)

2.6

5 S

5 S

5 S

(5)

0.0

29

22

18

(23)

5.6

20

17

18

(18)

1.5

9

7

9

(8)

1.2

150 µg

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

26

24

24

(25)

1.2

14 S

9 S

13 S

(12)

2.6

3 S

3 S

3 S

(3)

0.0

500 µg

N/T

N/T

21

19

27

(22)

4.2

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

1500 µg

N/T

N/T

11 S

25 S

19 S

(18)

7.0

N/T

N/T

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

725

564

568

(619)

91.8

1013

1559

1566

(1379)

317.3

678

753

617

(683)

68.1

228

155

208

(197)

37.7

196

163

260

(206)

49.3

Table 4: Test Results: Experiment 2 – With Metabolic Activation(Pre-Incubation)

Test Period

From: 02 November 2017

To: 05 November 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

(DMSO)

106

119

95

(107)

12.0#

19

16

10

(15)

4.6

32

34

26

(31)

4.2

28

28

32

(29)

2.3

12

14

14

(13)

1.2

0.15 µg

104

86

96

(95)

9.0

12

12

16

(13)

2.3

N/T

N/T

N/T

0.5 µg

115

114

103

(111)

6.7

15

11

10

(12)

2.6

N/T

30

25

24

(26)

3.2

18

17

9

(15)

4.9

1.5 µg

88

94

88

(90)

3.5

10

9

14

(11)

2.6

38

38

42

(39)

2.3

26

28

29

(28)

1.5

12

13

12

(12)

0.6

5 µg

108

100

91

(100)

8.5

9

12

11

(11)

1.5

29

30

38

(32)

4.9

23

22

20

(22)

1.5

10

13

14

(12)

2.1

15 µg

89

90

92

(90)

1.5

13

12

9

(11)

2.1

37

47

19

(34)

14.2

22

23

26

(24)

2.1

10

14

8

(11)

3.1

50 µg

81

82

59

(74)

13.0

9

8

14

(10)

3.2

31

35

27

(31)

4.0

23

18

29

(23)

5.5

12

10

9

(10)

1.5

150 µg

52 S

63 S

49 S

(55)

7.4

5 S

4 S

8 S

(6)

2.1

30

23

17

(23)

6.5

17

17

20

(18)

1.7

6

8

9

(8)

1.5

500 µg

N/T

N/T

23

23

23

(23)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

1500 µg

N/T

N/T

0 V

0 V

0 V

(0)

0.0

N/T

N/T

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

697

485

848

(677)

182.4

234

214

265

(238)

25.7

134

141

201

(159)

36.8

127

130

162

(140)

19.4

263

291

351

(302)

45.0

†: Experimental procedure repeated at a later date due to high colony counts in the original test

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

S: Sparse bacterial background lawn

T: Toxic, no bacterial background lawn

V: Very weak bacterial background lawn

#: Standard deviation

N/T: Not tested at this dose level

The maximum dose level of the test substance 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 substance induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9‑mix) initially from 150 µg/plate. Consequently, the toxic limit of the test substance was selected as the maximum dose level in the second mutation test.Results from the second mutation test showed that the test substance induced a stronger toxic response employing the pre‑incubation modification with weakened bacterial background lawns initially noted in the absence of S9‑mix from 15 µg/plate (TA100), 50 µg/plate (TA1535) and 150 µg/plate (TA98 and TA1537) and 1500 µg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100 and TA1535), 500 µg/plate (TA98 and TA1537) and 1500 µg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

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 biologically relevant 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).A small, statistically significant increase in TA1535 revertant colony frequency was observed in the presence of S9-mix at 15 µg/plate in the first mutation test. This increase, although in excess of the in-house historical control maxima, was considered to be of no biological relevance because there was no evidence of a dose‑response relationship and the fold increase was only 1.1 times the concurrent vehicle control.

Conclusions:
Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test with and without metabolic activation.
Executive summary:

A study was conducted to determine the genotoxic potential of the test substance, 'iso and anteiso C10-40 AAP EDM-ES' (active: 96%), according to the 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 test substance 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 metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate (1.5, 5, 15, 50, 150, 500, 1500 and 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 following the results of Experiment 1 and ranged between 0.15 and 1500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. Seven test substance concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the toxic limit of the test substance 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 substance 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 substance induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9 mix) initially from 150 µg/plate. Consequently, the toxic limit of the test substance was selected as the maximum dose level in the second mutation test. Results from the second mutation test showed that the test substance induced a stronger toxic response employing the pre incubation modification with weakened bacterial background lawns initially noted in the absence of S9 mix from 15 µg/plate (TA100), 50 µg/plate (TA1535) and 150 µg/plate (TA98 and TA1537) and 1500 µg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100 and TA1535), 500 µg/plate (TA98 and TA1537) and 1500 µg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9-mix and experimental methodology. 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 biologically relevant 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). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the presence of S9-mix at 15 µg/plate in the first mutation test. This increase, although in excess of the in-house historical control maxima, was considered to be of no biological relevance because there was no evidence of a dose response relationship and the fold increase was only 1.1 times the concurrent vehicle control. Under study conditions, the test substance, 'iso and anteiso C10-40 AAP EDM-ES' was considered to be non-mutagenic with and without metabolic activation (Envigo, 2017).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study was conducted to determine the genotoxic potential of the test substance, 'iso and anteiso C10-40 AAP EDM-ES' (active: 96%), according to the 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 test substance 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 metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate (1.5, 5, 15, 50, 150, 500, 1500 and 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 following the results of Experiment 1 and ranged between 0.15 and 1500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. Seven test substance concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the toxic limit of the test substance 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 substance 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 substance induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9 mix) initially from 150 µg/plate. Consequently, the toxic limit of the test substance was selected as the maximum dose level in the second mutation test. Results from the second mutation test showed that the test substance induced a stronger toxic response employing the pre incubation modification with weakened bacterial background lawns initially noted in the absence of S9 mix from 15 µg/plate (TA100), 50 µg/plate (TA1535) and 150 µg/plate (TA98 and TA1537) and 1500 µg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100 and TA1535), 500 µg/plate (TA98 and TA1537) and 1500 µg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9-mix and experimental methodology. 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 biologically relevant 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). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the presence of S9-mix at 15 µg/plate in the first mutation test. This increase, although in excess of the in-house historical control maxima, was considered to be of no biological relevance because there was no evidence of a dose response relationship and the fold increase was only 1.1 times the concurrent vehicle control. Under study conditions, the test substance, 'iso and anteiso C10-40 AAP EDM-ES' was considered to be non-mutagenic with and without metabolic activation (Envigo, 2017).

Justification for classification or non-classification

Based on the results of the in vitro Ames test, the test substance, 'iso and anteiso C10-40 AAP EDM-ES', does not warrant classification for genotoxicity according to the EU CLP criteria (Regulation 1272/2008/EC).​​