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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2014 to 29 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Benzoic acid, 2-hydroxy-,C14-18 alkyl derivs.
EC Number:
931-472-4
Cas Number:
182700-89-6
IUPAC Name:
Benzoic acid, 2-hydroxy-,C14-18 alkyl derivs.
Test material form:
liquid: viscous
Details on test material:
- Appearance: Clear reddish-brown viscous liquid
- Storage conditions of test material: At room temperature in the dark

Method

Target gene:
S. typhimurium: Histidine locus
E. coli: Tryptophan locus
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: Enriched nutrient broth (Oxoid LTD, Hampshire, England)
- Properly maintained: Yes. The 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 were stored in liquid nitrogen (-196 °C).
Additional strain / cell type characteristics:
other: Additional mutations: rfa: deep rough; gal: mutation in the galactose metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair system; and TA98 and TA100 possessed the R-factor = plasmid pKM101.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Enriched nutrient broth (Oxoid LTD, Hampshire, England)
- 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).
Additional strain / cell type characteristics:
other: Additional mutations: rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in the galactose metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; and uvrB.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
- Experiment 1: 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (all tester strains)
- Experiment 2 (without metabolic activation): 17, 52, 164, 512, 878, 1600 and 2800 µg/plate (all strains)
- Experiment 2 (with metabolic activation): 17, 52, 164, 512, 1600, 2800 and 5000 µg/plate (all strains)
- Experiments 3 and 4 (without metabolic activation): 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (TA98 only)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
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)
Top agar in top agar tubes was melted by heating to 45 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10⁹ 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 vortex mixed 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.

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Assessment of background lawn
Evaluation criteria:
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than 2 times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than 3 times the concurrent vehicle control.
b) The negative response should be reproducible in at least one a follow-up experiment.

A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than 2 times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than 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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In TA98 in Experiment 1 (- S9-mix) an up to 4.9-fold increase in the number of revertant colonies was observed at two intermediate dose levels but could not be replicated. This increase is considered to be incidental and not biologically relevant.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
not applicable
Positive controls validity:
valid
Additional information on results:
FIRST MUTATION EXPERIMENT
- Precipitate: Heavy precipitation of the test material on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate. The concurrent control plates with only the test material showed small round-off test material droplets (quite similar to microcolonies) at the highest dose level of 5000 μg/plate. Therefore the highest dose level was judged as not countable due to heavy precipitate.
- Toxicity: 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. The reduction of the bacterial background lawn and the reduction in the number of revertants are presented in Table 1.
- Mutagenicity: In tester strain TA98, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control in the absence of S9-mix. This increase was observed at two intermediate dose levels (52 and 164 μg/plate). In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material.

SECOND MUTATION ASSAY
- Precipitate: Precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 2800 and 5000 μg/plate in the presence of S9-mix. At the end of the incubation period precipitate was observed at concentrations of 2800 μg/plate in the absence of S9-mix and at 2800 and 5000 μg/plate in the presence of S9-mix in all tester strains. In the tester strain TA1537 in the absence of S9-mix, precipitate was already observed at the concentration of 1600 μg/plate. The concurrent control plates with only the test material showed small round-off test material droplets (quite similar to microcolonies) at the highest dose level of 5000 μg/plate. Therefore the highest dose level was judged as not countable due to heavy precipitate.
- Toxicity: The reduction of the bacterial background lawn and the reduction in the number of revertants are presented in Table 2.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

ADDITIONAL MUTATION ASSAY
To verify the mutagenic response observed in the first mutation experiment in the tester strain TA98 in the absence of S9-mix, two additional experiments (experiments 3 and 4) were performed.
- Precipitate: Precipitation of the test material on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate.
- Toxicity: An extreme thinning of the micro-colony lawn and an increase in the size of the microcolonies compared to the solvent control plate was observed in the third mutation experiment at test material concentrations of 512 and 1600 μg/plate and in the fourth experiment at 512 μg/plate. A complete lack of any micro-colony background lawn was observed in the third mutation experiment at the test material concentration of 5000 μg/plate and in the fourth experiment at 1600 and 5000 μg/plate.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
In the tester strain TA98 in the absence of S9-mix, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was observed only at two intermediate dose levels (52 and 164 μg/plate). Furthermore, this increase could not be repeated in three repeat experiments. Therefore, this increase is considered to be an incidental finding not biologically relevant.
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 two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Toxicity in the First Mutation Experiment (Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain

Without S-9 Mix

With S9-Mix

Dose (µg/plate)

Bacterial Background Lawn

Revertant Colonies

Dose (µg/plate)

Bacterial Background Lawn

Revertant Colonies

TA1535

512

1600

5000

Slight

Extreme

Absent

No reduction

Microcolonies

ND

5000

Normal

ND

TA1537

512

1600

5000

Slight

Extreme

Absent

Moderate

Microcolonies

ND

1600

5000

Normal

Normal

Extreme

ND

TA98

512

1600

5000

Slight

Extreme

Absent

Reduction*

Microcolonies

ND

5000

Normal

ND

TA100

52 to 164

512

1600

5000

Normal

Slight

Extreme

Absent

Extreme

Extreme

Microcolonies

ND

1600

5000

Normal

Normal

Moderate

ND

WP2uvrA

5000

Normal

ND

5000

Normal

ND

*Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range.

ND = Due to heavy precipitate, no revertant colonies could be determined

 

Table 2: Toxicity in the Second Mutation Experiment (Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain

Without S-9 Mix

With S9-Mix

Dose (µg/plate)

Bacterial Background Lawn

Revertant Colonies

Dose (µg/plate)

Bacterial Background Lawn

Revertant Colonies

TA1535

512

878

1600

2800

Slight

Slight

Extreme

Extreme

Moderate

No reduction

Microcolonies

Microcolonies

5000

Normal

ND

TA1537

164

512

878

1600

2800

Slight

Mod to ex*

Extreme

Extreme

Extreme

No reduction

Ex or micro**

Microcolonies

Microcolonies

Microcolonies

2800

5000

Normal

Normal

Extreme

ND

TA98

164

512

878

1600

2800

Slight

Moderate

Moderate

Extreme

Extreme

No reduction

No reduction

Extreme

Microcolonies

Microcolonies

5000

Normal

ND

TA100

512

878

1600

2800

Slight

Moderate

Extreme

Extreme

Moderate

Extreme

Microcolonies

Microcolonies

5000

Normal

ND

WP2uvrA

2800

Normal

No reduction

5000

Normal

ND

*Moderate to extreme

**Extreme or microcolonies

ND = Due to heavy precipitate, no revertant colonies could be determined

Key to Toxicity Tables

Bacterial background lawn evaluation

- Normal: Distinguished by a healthy microcolony lawn

- Slightly reduced: Distinguished by a slight thinning of the microcolony lawn

- Moderately reduced: Distinguished by a moderate thinning of the microcolony lawn

- Extremely reduced: Distinguished by an extreme thinning of the microcolony lawn and an increase in the size of the microcolonies compared to the solvent control plate

- Absent: Distinguished by a complete lack of any microcolony background lawn

 

Evaluation of the reduction in the number of revertants

- A reduction of 21 to 40 %: slight reduction

- A reduction of 41 to 60 %: moderate reduction

- A reduction of 61 to 99 %: extreme reduction

If the size of the microcolonies was increased to small colonies due to an extremely reduced background lawn the reduction is evaluated as microcolonies. If no revertant colonies are observed on the plates the reduction is evaluated as a complete lack of revertants.

Any mean plate count equal to the minimal value of the historical control data range should be considered not toxic.

Table 3: Summary of Experiment 1

+/- 5 % S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

5.4

17

52

164

512

1600

5000

88

112

92

16

15

34

MC

HP

20

19

20

12

13

15

MC

HP

29

28

25

23

24

19

24

HP

16

11

20

78

54

8

MC

HP

5

8

10

5

4

2

MC

HP

+

Solvent

5.4

17

52

164

512

1600

5000

103

87

90

83

84

76

56

HP

11

11

10

10

12

14

10

HP

30

34

34

26

21

32

31

HP

26

29

29

27

25

18

14

HP

12

7

9

8

8

4

2

HP

Positive Controls

-

Name

MMS

SA

4NQO

NF

ICR-191

Concentration (µg/plate)

650

5

10

10

2.5

Mean no. colonies/plate

549

728

1087

699

729

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

1

2.5

15

1

2.5

Mean no. colonies/plate

806

174

158

756

320

MC = Microcolonies and an extremely reduced background lawn were seen

HP = Heavy precipitate. The number of revertant colonies could not be determined

MMS = Methylmethanesulfonate

SA = Sodium azide

4NQO = 4-Nitroquinoline-N-oxide

NF = 2-Nitrofluorene

2AA = 2-aminoanthracene

 

Table 2: Summary of Experiment 2

+/- 10 % S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

17

52

164

512

878

1600

2800

103

95

94

74

49

29

MC

MC

11

15

13

13

5

7

MC

MC

38

26

34

20

27

26

22

17

8

14

15

13

8

2

MC

MC

5

8

6

7

1

MC

MC

MC

+

Solvent

17

52

164

512

1600

2800

5000

98

103

98

92

87

89

71

HP

14

12

12

12

9

10

9

HP

38

34

40

41

43

39

29

HP

21

21

24

20

18

16

22

HP

10

6

9

7

13

3

2

HP

Positive Controls

-

Name

MMS

SA

4NQO

NF

ICR-191

Concentration (µg/plate)

650

5

10

10

2.5

Mean no. colonies/plate

821

639

1285

933

844

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

2

2.5

15

1

5

Mean no. colonies/plate

1360

190

309

500

387

MC = Microcolonies and an extremely reduced background lawn were seen

HP = Heavy precipitate. The number of revertant colonies could not be determined

MMS = Methylmethanesulfonate

SA = Sodium azide

4NQO = 4-Nitroquinoline-N-oxide

NF = 2-Nitrofluorene

2AA = 2-aminoanthracene

 

Table 5: Summary of Experiments 3 and 4

+/- S9 Mix

Concentration

(µg/plate)

Experiment 3

Experiment 4

TA98

TA98

-

Solvent

17

52

164

512

878

1600

2800

19

18

13

18

17

MC

MC/HP

HP

12

15

16

12

15

MC

MC

MC/HP

NF 10

984

1224

MC = Microcolonies and an extremely reduced background lawn were seen

HP = Heavy precipitate. The number of revertant colonies could not be determined

NF = 2-Nitrofluorene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.
Executive summary:

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and 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). To obtain more information about the possible mutagenicity of the test material, additional experiments were performed with the tester strain TA98 in the absence of S9-mix.

In the first mutation assay, the test material was tested up to the concentration of 5000 μg/plate in DMSO in the absence and presence of 5 % (v/v) S9-mix. The test material precipitated heavily on the plates at the dose level of 5000 μg/plate and due to this heavy precipitate, the number of revertants at this dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in TA1537 and TA100 in the presence of S9-mix. In tester strain TA98, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control in the absence of S9-mix. This increase was observed at two intermediate dose levels (52 and 164 μg/plate). In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material.

In the second mutation assay, the test material was tested up to the concentration of 2800 μg/plate in the absence of S9-mix and up to 5000 μg/plate in the presence of 10 % (v/v) S9-mix. The test material precipitated on the plates at dose levels of 2800 μg/plate and above. In the tester strain TA1537 in the absence of S9-mix, precipitate was already observed at the concentration of 1600 μg/plate. Due to the heavy precipitate on the plates at the test material concentration of 5000 μg/plate, the number of revertants at this dose level could not be determined. Cytotoxicity was observed in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in TA1537 in the presence of S9-mix. No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

To verify the mutagenic response observed in the first mutation experiment in the tester strain TA98 in the absence of S9-mix, two additional experiments (experiments 3 and 4) were performed. The test material was tested up to the concentration of 5000 μg/plate in the absence of S9-mix. The test material precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in the third mutation experiment at dose levels of 164 μg/plate and above and in the fourth mutation experiment at dose levels of 512 μg/plate and above. No increase in the number of revertants was observed upon treatment with the test material under all conditions tested

In the tester strain TA98 in the absence of S9-mix in the first experiment, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was observed only at two intermediate dose levels (52 and 164 μg/plate). Furthermore, this increase could not be repeated in three repeat experiments. Therefore, this increase is considered to be an incidental finding and not biologically relevant.

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 two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.