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Toxicological information

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:
26 May 2015 to 18 June 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:
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: Slightly yellow powder with lumps
- Storage conditions of test material: At room temperature

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):
- Stock cultures were stored in liquid nitrogen (-196 °C)
- Regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants
- 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. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilisation of the strain using Tris-EDTA treatment. 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:
- Preliminary test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (TA100 and WP2uvrA)
- Main study Experiments 1 and 2 (with and without metabolic activation): 17, 52, 164, 512 and 1600 µg/plate (TA1535, TA1537 and TA98)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
congo red
methylmethanesulfonate
other: 2-aminoanthracene, ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 2: pre-incubation

- Experiment 1
The dose range finding study with two tester strains is reported as a part of the direct plate assay.
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 (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 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 were counted.

- Experiment 2
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were pre-incubated for 30 minutes by 70 rpm at 37 °C, 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), 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. After the pre-incubation period the solutions were added to 3 mL molten top agar. 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 were counted.

DURATION
- Exposure duration: 48 ± 4 h

NUMBER OF REPLICATIONS: 3

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 observed.

OTHER
- 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.
Evaluation criteria:
- A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in 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 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 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 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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
Cytotoxicity / choice of top concentrations:
other: Only observed in tester strains TA1535 and TA1537 in the absence of S9-mix at the highest tested concentration (second experiment)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 examined
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING TEST/FIRST EXPERIMENT: DIRECT PLATE ASSAY
- Precipitate
Precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 1600 μg/plate and above at the end of the incubation period.

- Toxicity
There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
In tester strain TA98, a fluctuation in the mean number of revertant colonies below the laboratory historical control data range was observed at the mid dose of 512 μg/plate (presence of S9-mix). Since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test material; rather it is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

- Mutagenicity
No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

SECOND EXPERIMENT: PRE-INCUBATION ASSAY
- Precipitate
Precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 1600 μg/plate at the end of the incubation period.

- Toxicity
Cytotoxicity was only observed in the tester strains TA1535 and TA1537 in the absence of S9-mix, where moderate reductions of the revertant colonies were observed at the test material concentration of 1600 μg/plate in the absence of S9-mix. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix.
In tester strain TA1535, a fluctuation in the mean number of revertant colonies below the laboratory historical control data range was observed at the low dose of 52 μg/plate (absence of S9-mix). Since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test material; rather it is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.
In tester strain TA100, fluctuations in the mean number of revertant colonies below the laboratory historical control data range were observed in the presence of S9-mix. However, since no reductions below the concurrent vehicle control were seen, this reduction is not considered to be caused by toxicity of the test material, rather it is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

- Mutagenicity
No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of Experiment 1

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

1.7

5.4

17

52

164

512

1600

5000

103

113

108

114

92

96

104

85

99

12

-

-

6

13

11

12

6

-

28

26

27

27

29

30

30

24

23

17

-

-

19

10

14

15

7

-

5

-

-

6

7

11

10

4

-

+

Solvent

1.7

5.4

17

52

164

512

1600

5000

108

106

108

116

97

98

105

89

111

10

-

-

8

11

8

10

8

-

33

36

52

37

34

35

35

31

32

24

-

-

25

18

24

9

13

-

8

-

-

11

6

10

4

6

-

Positive Controls

-

Name

MMS

SA

4-NQO

NF

ICR-191

Concentration (µg/plate)

650

5

10

10

2.5

Mean no. colonies/plate

776

760

1326

1340

339

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

1

2.5

15

1

2.5

Mean no. colonies/plate

1389

275

218

908

324

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline N-oxide

NF = 2-nitrofluorene

2AA = 2-aminoanthracene

Table 2: Summary of Experiment 2 

± 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

1600

86

91

79

90

89

81

9

6

5

9

17

4

24

21

26

30

23

28

10

11

11

12

11

8

4

5

5

4

7

2

+

Solvent

17

52

164

512

1600

62

73

64

68

65

86

7

4

11

8

6

8

51

50

47

61

44

58

15

17

12

11

19

15

6

6

7

8

3

5

Positive Controls

-

Name

MMS

SA

4-NQO

NF

NF

Concentration (µg/plate)

650

5

10

10

15

Mean no. colonies/plate

699

72

153

1964

62

+

Name

2AA

2AA

2AA

CR

2AA

Concentration (µg/plate)

5

2.5

15

500

2.5

Mean no. colonies/plate

875

775

341

627

147

MMS = methylmethanesulfonate

SA = sodium azide

NF = 2-nitrofluorene

4-NQO = 4-nitroquinoline N-oxide

2AA = 2-aminoanthracene

CR = Congo red

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 dissolved in dimethyl sulfoxide 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 metabolic activation (rat liver S9-mix induced by Aroclor 1254). Experiment 1 was performed using the plate incorporation method and Experiment 2 was performed using the pre-incubation method.

A dose range finding study was performed as part of Experiment 1; the test material was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test material precipitated on the plates at dose levels of 1600 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the rest of the first mutation experiment, the test material was tested up to concentrations of 1600 μg/plate in the strains TA1535, TA1537 and TA98. The test material precipitated on the plates at the top dose of 1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test material was tested up to concentrations of 1600 μg/plate in all tester strains in the pre-incubation assay. The test material precipitated on the plates at the top dose of 1600 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the absence of S9-mix at the highest tested concentration.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 Acceptable responses were obtained for the negative and positive control materials, indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

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