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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:
01-02-2000 to 21-02-2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable deviations.
Remarks:
The efficacy of the S9-mix was only validated using 2-Anthramine (2-Aminoanthracene CAS 613-13-8) and benzopyrene or dimethylbenzanthracene was not used in each test. This is reflected in the reliability score.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The efficacy of the S9-mix was only validated using 2-Anthramine (2-Aminoanthracene CAS 613-13-8) and benzopyrene or dimethylbenzanthracene was not used in each test. This is reflected in the reliability score.
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry, 09 December 1986
Deviations:
yes
Remarks:
see above
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
see above
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
see above
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: September 1999 ; signature: December 1999
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: At room temperature, protected from light and under nitrogen gas
- Other: colourless

Method

Target gene:
histidine or tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test (TA98, TA100 and Wp2urvA): 0, 10, 100, 500, 1000, 2500 and 5000 µg/plate with and without S9 mix
Experiment 1 (plate incorporation method): All strains: 0, 312.5, 625, 1250, 2500, 5000 µg/plate with and without S9 mix
Experiment 2 (preincubation method): All strains: 0, 312.5, 625, 1250, 2500, 5000 µg/plate with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (batch number and supplier listed in full study report).
- Justification for choice of solvent/vehicle: Ethanol was selected as the vehicle. Test item was dissolved in the vehicle at a concentration of 100 mg/ml for the preliminary toxicity test and for both mutagenicity experiments. The preparations were made immediately before use.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preliminary Toxicity Test: in medium; in agar (plate incorporation), Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Preliminary Toxicity Test: To assess the toxicity of the test substance to the bacteria, at least six dose-levels (one plate/dose-level) will be tested in the TA98, TA100 and WP2uvrA strains, with and without S9 mix. Test item solution (0.05 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
Experiment 1. Test item solution (0.05 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
Experiment 2. Test item solution (0.05 mL), S9 mix (0.5 mL) and bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours incubation at 37 ºC the revertants were scored using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Evidence leading to positive result:
1. Reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level
2. Evidence of dose-relationship, in any strain
Reference to historical data and/or other considerations such as biological relevance would be taken into consideration during the evaluation of data

Applicant assessment indicates: The test item is considered non-mutagenic (negative) in the test system if the above criteria are not met. Instances of data prohibiting definitive judgement about test item activity may be reported as equivocal.

Acceptance Criteria:
The study is considered valid if the following criteria are met:
1. The number of revertants in the vehicle controls is consistent with the historical control data range (information attached to the full study report)
2. The number of revertants in the positive control is higher than the vehicle controls and consistent with the historical control range (information attached to the full study report).

Results and discussion

Test resultsopen allclose all
Species / strain:
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
Remarks:
See table 1 and 2, without S9 mix activation, slight to moderate cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
See table 1 and 2, without S9 mix activation, slight to moderate cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Table 1 : Test Results: Experiment 1 – Preliminary Toxicity Test : with and without metabolic activation and results of concurrent positive controls

Strains

Doses µg/plate

Without S9 Mix

With S9 Mix

 

 

T

E

Revertants per plate

T

E

Revertants per plate

TA98

0

0

0

24

0

0

29

 

10

0

0

21

0

0

24

 

100

0

0

19

0

0

23

 

500

0

0

14

0

0

17

 

1000

0

0

19

0

0

38

 

2500

0

1

23

0

1

20

 

5000

0

2

10

0

1

20

 

 

 

 

 

 

 

 

TA100

0

0

0

82

0

0

95

 

10

0

0

71

0

0

104

 

100

0

0

61

0

0

84

 

500

0

0

43

0

0

83

 

1000

1

0

55

0

0

82

 

2500

1

1

65

0

1

90

 

5000

1

1

38

0

1

90

 

 

 

 

 

 

 

 

WP2 uvrA

0

0

0

29

0

0

40

 

10

0

0

18

0

0

40

 

100

0

0

22

0

0

30

 

500

0

0

31

0

0

30

 

1000

0

0

25

0

0

22

 

2500

0

1

28

0

1

31

 

5000

0

1

21

0

1

34

 

 

 

 

 

 

 

 

0 = vehicle control (Ethanol)

Where:

T = toxicity : score = 0, none, 1 = slight, 2 = moderate, 3 = considerable to total

E = Emulsion of test item: score = 0, none, 1 = slight, 2 = moderate, 3 = strong

 

Table 2 : Test Results: Experiment 2 – Main Test (pre-incubation method) : with and without metabolic activation and results of concurrent positive controls

With S9 Mix

Strains

Doses µg/plate

T

E

Revertants per plate

Mean

Standard Deviation

 

 

 

 

1

2

3

 

 

TA1535

0

0

0

29

30

26

28

2

 

312.5

0

0

25

13

30

23

9

 

625

0

0

23

24

27

25

2

 

1250

0

0

21

13

41

25

14

 

2500

0

0

20

31

25

25

6

 

5000

0

1

27

29

30

29

2

 

2AM(2)

-

-

258

232

226

239

17

 

 

 

 

 

 

 

 

 

TA1537

0

0

0

6

10

7

8

2

 

312.5

0

0

8

4

8

7

2

 

625

0

0

13

7

7

9

3

 

1250

0

0

4

9

4

6

3

 

2500

0

0

15

8

7

10

4

 

5000

0

1

6

3

5

5

2

 

2AM(2)

-

-

81

75

98

85

12

 

 

 

 

 

 

 

 

 

TA98

0

0

0

19

12

21

17

5

 

312.5

0

0

14

15

12

14

2

 

625

0

0

28

13

16

19

8

 

1250

0

0

21

16

17

18

3

 

2500

0

0

28

13

14

18

8

 

5000

0

1

24

12

18

18

6

 

2AM(2)

-

-

1362

1317

1383

1354

34

 

 

 

 

 

 

 

 

 

TA100

0

0

0

139

132

114

128

13

 

312.5

0

0

92

81

93

89

7

 

625

0

0

93

111

91

98

11

 

1250

0

0

76

95

72

81

12

 

2500

0

0

80

74

77

77

3

 

5000

0

1

65

72

64

67

4

 

2AM(2)

-

-

1087

992

921

1000

83

 

 

 

 

 

 

 

 

 

WP2 uvrA

0

0

0

55

44

37

45

9

 

312.5

0

0

46

35

32

38

7

 

625

0

0

50

40

34

41

8

 

1250

0

0

39

26

44

36

9

 

2500

0

0

49

42

46

46

4

 

5000

0

1

39

32

38

36

4

 

2AM(10)

-

-

233

170

179

194

34

 

 

 

 

 

 

 

 

 

Without S9 Mix

TA1535

0

0

0

6

10

12

9

3

 

312.5

0

0

5

5

5

5

0

 

625

0

0

7

8

6

7

1

 

1250

0

0

7

7

5

6

1

 

2500

0

0

13

7

7

9

3

 

5000

0

1

7

10

8

8

2

 

NaN3(1)

-

-

646

616

703

655

44

 

 

 

 

 

 

 

 

 

TA1537

0

0

0

9

14

13

12

3

 

312.5

1

0

5

5

5

5

0

 

625

1

0

8

10

6

8

2

 

1250

2

0

11

7

8

9

2

 

2500

2

0

11

7

7

8

2

 

5000

2

1

9

6

5

7

2

 

9AA(50)

-

-

332

287

435

351

76

 

 

 

 

 

 

 

 

 

TA98

0

0

0

17

16

15

16

1

 

312.5

0

0

11

13

10

11

2

 

625

0

0

18

10

14

14

4

 

1250

1

0

12

17

17

15

3

 

2500

1

0

22

14

18

18

4

 

5000

2

1

15

20

10

15

5

 

2NF(0.5)

-

-

150

164

147

154

9

 

 

 

 

 

 

 

 

 

TA100

0

0

0

107

97

93

99

7

 

312.5

0

0

94

80

81

85

8

 

625

0

0

124

102

104

110

12

 

1250

0

0

83

77

88

83

6

 

2500

0

0

119

90

96

102

15

 

5000

0

1

106

96

76

93

15

 

NaN3(1)

-

-

793

771

773

779

12

 

 

 

 

 

 

 

 

 

WP2 uvrA

0

0

0

30

20

34

28

7

 

312.5

0

0

27

24

28

26

2

 

625

0

0

31

40

32

34

5

 

1250

0

0

35

27

28

30

4

 

2500

0

0

40

29

27

32

7

 

5000

0

1

27

25

23

25

2

 

4NQQ(2)

-

-

1761

1781

1791

1778

15

 

 

 

 

 

 

 

 

 

0 = vehicle control (Ethanol)

Where:

T = toxicity : score = 0, none, 1 = slight, 2 = moderate, 3 = considerable to total

E = Emulsion of test item: score = 0, none, 1 = slight, 2 = moderate, 3 = strong

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD Guideline 471, Japanese guidelines for bacterial mutagenicity testing and EU Method B.14, under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix activation system. The test strains were treated in two independent experiments with the test item, utilising the Ames plate incorporation method and then preincubation method, respectively at up to five dose levels, in triplicate, both with and without the addition of a rat liver microsomal metabolic activation system (10% liver S9). The vehicle was ethanol. The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The Experiment 1 dose levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for all Salmonella strains: TA98, TA100, TA1535, TA1537 and for E.coli strain WP2uvrA-. The experiment was repeated using the same dose range by the pre-incubation method in Experiment 2. All of the positive controls used in the test induced expected increases in the frequency of revertant colonies, both with or without metabolic activation. The vehicle controls were considered acceptable. Therefore the assay was considered valid. The maximum dose level of the test item in the Experiment 1 and Experiment 2 was the maximum recommended dose level of 5000 μg/plate in all tester strains. A slight emulsion was observed in the plates when scoring the revertants mainly at the highest dose-level. Without S9 mix, a slight to moderate toxicity was generally induced, depending on the dose-levels and the tester strains. With S9 mix, except for a slight decrease in the number of revertants, noted in the second experiment (preincubation method) in the TA1537 and TA100 strains at the highest dose-level, no noteworthy toxicity was induced in both experiments. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.