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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July 2016 to 16 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
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. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl 4-(dimethylamino)benzoate
EC Number:
244-289-3
EC Name:
2-ethylhexyl 4-(dimethylamino)benzoate
Cas Number:
21245-02-3
Molecular formula:
C17H27NO2
IUPAC Name:
2-ethylhexyl 4-(dimethylamino)benzoate
Test material form:
liquid
Details on test material:
- Appearance: Pale yellow liquid
- Storage conditions of test material: Room temperature in the dark
- Expiry date: 19 November 2017

Method

Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon)
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and identity of media: Overnight subcultures of stock cultures were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours.
- Properly maintained: Yes, each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). All of the strains were stored at approximately -196 °C in a iquid nitrogen freezer.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and identity of media: Overnight subcultures of stock cultures were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours.
- Properly maintained: Yes, each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. Prior to use, the master strain was checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). All of the strains were stored at approximately -196 °C in a iquid nitrogen freezer.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
- Experiment 1 (with and without metabolic activation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate)
- Experiment 2 (with and without metabolic activation): 15, 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulphoxide)
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controls
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1; pre-incubation for Experiment 2

EXPERIMENT 1: PLATE INCORPORATION METHOD
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 either 0.5 mL of S9-mix or phosphate buffer (for assays with and without metabolic activation, respectively). These were then mixed and overlayed onto a Vogel-Bonner minimal agar plate. Negative (untreated) controls were also performed on the same day as the mutation test.
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. Manual counts were performed at 5000 μg/plate because of test material precipitation. Several further manual counts were also required due to revertant colonies spreading slightly, thus distorting the actual plate count.

EXPERIMENT 2: PRE-INCUBATION METHOD
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of S9-mix or phosphate buffer (for assays with and without metabolic activation, respectively) 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 minimal agar plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method.
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. Manual counts were performed at 5000 μg/plate because of test material precipitation.

NUMBER OF REPLICATIONS: Triplicate assays were performed

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the growth of the bacterial background lawn and increases in the frequency of revertant colonies were recorded to determine cytotoxicity. The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
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 presented as follows: TA1535: 7 to 40; TA100: 60 to 200; TA1537: 2 to 30; TA98: 8 to 60; 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 item 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:
1. A dose-related increase in mutant frequency over the dose range tested.
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).
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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 and Table 2 for Experiment 1 and Table 3 and Table 4 for Experiment 2.
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. 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.
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). A test material precipitate (cream-coloured and particulate in appearance) was noted at and above 1500 μg/plate; this observation did not prevent the scoring of revertant colonies.
There were no significant 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 toxicologically 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). A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.
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.

Any other information on results incl. tables

Table 1: Number of revertants (mean) ± SD in Experiment 1- without metabolic activation

Dose Level per plate (µg)

TA100

TA1535

WP2uvrA

TA98

TA1537

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

65

1.2

11

4.6

21

4.5

19

1.7

13

3.6

1.5

63

2.3

10

2.3

21

5.5

20

1.5

18

6.8

5

64

0.6

9

1.7

26

9.7

14

6.0

16

4.6

15

67

0.6

15

4.6

24

8.2

18

4.7

15

4.5

50

65

1.5

10

0.6

19

6.8

19

1.5

11

3.2

150

68

4.9

9

2.0

23

8.1

21

4.0

11

2.9

500

68

5.3

11

4.6

19

10.7

18

6.4

8

1.5

1500

62P

1.0

10P

1.5

18P

2.3

20P

0.6

13P

0.0

5000

62P

1.0

11P

2.6

13P

5.5

17P

5.0

9P

3.2

Positive Controls (µg)

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

536

28.3

591

69.7

856

52.1

267

1.7

313

25.1

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

P = Precipitate

Table 2: Number of revertants (mean) ± SD in Experiment 1- with metabolic activation

Dose Level per plate (µg)

TA100

TA1535

WP2uvrA

TA98

TA1537

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

75

11.5

12

2.3

18

4.7

30

8.7

18

5.5

1.5

69

13.3

13

4.4

24

5.8

28

3.1

18

4.5

5

79

6.1

9

0.0

19

3.2

28

4.4

13

5.9

15

71

9.6

11

1.0

23

0.6

34

11.2

10

1.7

50

70

12.4

11

0.6

23

3.5

27

8.7

12

2.1

150

76

4.4

15

5.0

18

2.1

33

6.7

9

1.0

500

81

8.0

10

1.0

20

3.8

35

1.7

11

7.8

1500

70P

3.2

10P

1.2

21P

2.3

26P

0.6

13P

1.2

5000

66P

3.8

8P

0.6

19P

2.5

24P

3.8

11P

2.3

Positive Controls (µg)

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1119

64.1

257

9.0

460

70.7

128

5.5

261

12.1

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

P = Precipitate

 

Table 3: Number of revertants (mean) ± SD in Experiment 2- without metabolic activation

Dose Level per plate (µg)

TA100

TA1535

WP2uvrA

TA98

TA1537

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

91

6.1

17

7.6

33

7.2

26

6.2

15

6.9

15

76

5.8

18

6.1

27

1.0

25

5.0

11

1.2

50

95

4.0

18

8.7

30

12.0

27

6.1

14

1.5

150

108*

12.1

21

10.7

28

12.0

19

4.0

16

3.1

500

99

1.5

13

1.7

27

6.1

24

5.2

20

2.5

1500

89P

3.1

14P

4.0

29P

0.6

18P

4.0

21P

1.2

5000

77P

4.2

11P

3.0

20P

3.2

19P

0.6

9P

1.7

Positive Controls (µg)

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

969

346.8

1862

173.4

1025

65.0

283

7.1

301

126.7

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

*p≤0.05

P = Precipitate

Table 4: Number of revertants (mean) ± SD in Experiment 2- with metabolic activation

Dose Level per plate (µg)

TA100

TA1535

WP2uvrA

TA98

TA1537

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

91

1.2

17

4.2

31

6.0

24

2.1

17

8.5

15

87

13.5

18

5.1

28

5.0

26

3.8

22

4.6

50

108

13.5

13

2.9

31

6.4

32

4.7

19

1.0

150

101

12.1

13

2.6

33

2.9

26

7.9

20

4.2

500

96

9.9

10

2.6

21

10.7

30

5.1

17

4.0

1500

103P

6.1

12P

2.1

26P

8.1

29P

9.5

22P

4.0

5000

73P

5.9

10P

1.5

22P

7.0

26P

4.6

11P

2.0

Positive Controls (µg)

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1173

25.2

181

12.9

195

16.7

108

14.4

129

3.5

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

P = Precipitate

Applicant's summary and conclusion

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

A bacterial reverse mutation assay was carried out in accordance with the standardised guidelines OECD 471, EU Method B13/14, US EPA OCSPP 870.5100 and the testing published by the Japanese METI, MHLW and JMAFF under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material in DMSO 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 15 to 5000 μg/plate. Bacteria were also exposed to vehicle controls and appropriate positive controls.

The vehicle 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.

In Experiment 1 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). 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). A test material precipitate (cream-coloured and particulate in appearance) was noted at and above 1500 μg/plate; this observation did not prevent the scoring of revertant colonies.

There were no significant 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 in Experiment 1. Similarly, no toxicologically 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 in Experiment 2. A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain.

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

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