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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 2016 to 18 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:
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Yellow powder
- Storage conditions of test material: At room temperature protected from light
- Test material handling: Use amber glassware or wrap container in aluminium foil
- Stable at higher temperatures: Yes, maximum temperature: 95 °C

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):
- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver S9-mix induced by Aroclor 1254)
Test concentrations with justification for top dose:
- Dose range finding study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 1 (TA1535, TA1537 and TA98): 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 2 (all strains): 492, 878, 1568, 2800 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 3 (TA98 only): 500, 1000, 2000, 3000, 4000 and 5000 (absence and presence of S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed. The test material was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves until the test material had completely dissolved.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DOSE RANGE FINDING TEST/ MUTATION ASSAY
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5 % (v/v) S9-mix and reported as part of the first mutation experiment.

MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test material were tested in each strain both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains. An additional experiment was performed with TA98.
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
- Precipitate: Precipitation on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain TA1537 in the absence of S9-mix.
In the tester strains TA1537 and TA98, both in the absence of S9-mix, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test material. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
- Mutagenicity: In tester strain TA98, the test material induced an up to 4.1-fold increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material.

MUTATION EXPERIMENT 2
- Precipitate: Precipitation on the plates was not observed at the start or at the end of the incubation period.
- Toxicity: Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix.
- Mutagenicity: In tester strain TA98, the test material induced an up to 3.1-fold increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material.
In strain TA100, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the absence of S9-mix at the mid-dose level of 878 μg/plate. However, since the increase was at the mid-dose level, not dose related and caused by one outlier, this increase was not considered to be biologically relevant.

MUTATION EXPERIMENT 3
- Precipitate: Precipitation of on the plates was not observed at the start or at the end of the incubation period.
- Toxicity: Cytotoxicity was observed in the absence and presence of S9-mix.
- Mutagenicity: No biologically relevant increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
In the presence of S9-mix, 4.1- and 3.1-fold increases were observed in tester strain TA98 in the first and second mutation experiment, respectively. These responses could not be repeated in the additional third experiment, a maximum of 2.1-fold was reached. Furthermore, the 4.1-fold increase observed in the first mutation experiment was related to a low (9 revertants/plate versus historical mean of 25 revertants/plate) solvent control value and was not above laboratory historical control data range. The 3.1-fold increase observed in the second mutation experiment was just above laboratory historical control data range, but not dose related. Therefore, these increases are considered to be not biologically relevant.
In the presence of S9-mix, all other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the experiments.
The negative control values were within the laboratory historical control data ranges, except the response for TA1537 (presence of S9-mix) and TA98 (absence of S9-mix), first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (2 and 8 revertant colonies) when compared against relevant historical control data (3 and 9 relevant colonies), the validity of the test was considered to be not affected.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table 1: Dose Range-finder and Experiment 1 (Plate incorporation assay 5 % S9)

+/- S9 Mix

Concentration (µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

PC

DMSO

1.7

5.4

17

52

164

512

1600

5000

1123

102

127

118

100

106

113

127

116 n

42 s NP

820

6

-

-

-

12

6

11 n

9 s

5 m NP

1306

25

26

25

24

22

20

23

24 n

15 s NP

1126

8

-

-

-

15

8

8

15 n

5 s NP

1017

5

-

-

-

2

1

5

8

6 n NP

+

PC

DMSO

1.7

5.4

17

52

164

512

1600

5000

1326

110

96

113

100

108

111

124 n

95 s

57 m NP

232

12

-

-

-

6

9

13 n

5 s

3 m NP

390

23

34

40

29

28

28

28

31 n

18 m NP

690

9

-

-

-

16

17

26

37 n

26 m NP

242

2

-

-

-

5

7

4

5 n

4 s NP

Mean number of revertant colonies/3 replicate plates

PC = Positive control

NP = No precipitate

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

m = Bacterial background lawn moderately reduced

 

Table 2: Experiment 2 (Plate incorporation assay 10 % S9)

+/- S9 Mix

Concentration (µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

PC

DMSO

492

878

1568

2800

5000

978

94

125

190

114 n

78 s

27 m NP

840

11

14

18

19 n

2 s

5 m NP

1192

24

25

35

34 n

31 s

21 m NP

1238

9

10

21

8

7 n

1 m NP

1064

9

10

11

15 n

2 s

2 m NP

+

PC

DMSO

492

878

1568

2800

5000

1300

80

102

125

70 n

54 s

26 m NP

141

7

17

19

11 n

4 s

5 m NP

384

37

25

25

34

29 n

11 m NP

602

19

24

32

26

58 n

36 m NP

449

11

9

8

10 n

5 s

3 m NP

Mean number of revertant colonies/3 replicate plates

PC = Positive control

NP = No precipitate

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

m = Bacterial background lawn moderately reduced

 

Table 3: Experiment 3 (Plate incorporation assay 10 % S9)

+/- S9 Mix

Concentration (µg/plate)

Mean number of colonies/plate

Frameshift Type

TA98

-

PC

DMSO

492

878

1568

2800

5000

1559

18

11 n

16 s

25 s

13 s

6 s

4 m NP

+

PC

DMSO

492

878

1568

2800

5000

811

21

31

33

45 n

39 s

34 s

30 m NP

Mean number of revertant colonies/3 replicate plates

PC = Positive control

NP = No precipitate

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

m = Bacterial background lawn moderately reduced

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). An additional experiment was performed with tester strain TA98.

In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in both tester strains in the absence and presence of S9-mix. Results of this dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test material was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains, with the exception of tester strain TA1537 in the absence of S9-mix.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10 % (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was observed in all tester strains.

To verify the increase in the number of revertant colonies in tester strain TA98 an additional experiment was performed. In this third mutation experiment, the test material was tested at a concentration range of 500 to 5000 μg/plate in the absence and presence of 10 % (v/v) S9-mix in tester strain TA98. Toxicity was observed in the absence and presence of S9-mix.

In the absence of S9-mix, all bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the experiments.

In the presence of S9-mix, 4.1- and 3.1-fold increases were observed in tester strain TA98 in the first and second mutation experiments, respectively. These responses could not be repeated in the additional third experiment, a maximum of 2.1-fold was reached. Furthermore, the 4.1-fold increase observed in the first mutation experiment was related to a low (9 revertants/plate versus historical mean of 25 revertants/plate) solvent control value and was not above laboratory historical control data range. The 3.1-fold increase observed in the second mutation experiment was just above laboratory historical control data range, but not dose related. Taken together, these increases are considered to be not biologically relevant.

In the presence of S9-mix, all other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the experiments.

In this study, acceptable responses were obtained for the negative and strain-specific positive control materials indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.