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Genetic toxicity: in vitro

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Administrative data

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:
1 August 1995 to 18 August 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted 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. The study report was conclusive, performed to valid guidelines and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

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:
- Name of test material (as cited in study report): M-377
- Physical state: solid
- Appearance: garnet powder
- Storage condition of test material: room temperature and protected from light

Method

Target gene:
Histidine synthesis
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: rfa (all strains), uvr B (all strains), pKM 101 (TA98, TA 100)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Species / strain:
S. typhimurium TA 102
Additional strain characteristics:
other: rfa, pKM 101, pAQ1
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary test:
0, 10, 100, 500, 1000, 2500, 5000 μg/plate (stains; TA 98, TA 100 and TA 102, with and without metabolic activation)

First Mutagenicity test:
0, 125, 250, 500, 1000, 2000 μg/plate (strains; TA 1535, TA 1537, TA 100 and TA 102, with and without metabolic activation)
0, 114.5, 229, 458, 916, 1832 μg/plate (strain; TA 98, with and without metabolic activation)

Second Mutagenicity test:
0, 125, 250, 500, 1000, 2000 μg/plate (stains; TA 1535, TA 1537, TA 98, TA 100 and TA 102; with and without metabolic activation)
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
mitomycin C
other: 2-anthramine
Details on test system and conditions:
STUDY DESIGN: A preliminary test, followed by two independent mutagenicity tests.

METHOD OF APPLICATION: preincubation
The day before treatment, cultures were inoculated from frozen permanents. A crystal was sampled under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours.
0.05 to 0.1 mL of the test material solution, 0.5 mL phosphate buffer pH 7.4 (without S9 mix) or 0.5 mL of S9 mix (with S9 mix) and 0.1 mL of the strain were incubated for 30 minutes at 30 °C prior to adding the overlay agar and pouring onto the surface of a minimal agar plate (the agar containing 0.5 % rather than 2 % glucose).

DURATION
- Exposure duration: Plates were incubated inverted at 37 °C for 48 to 72 hours.

NUMBER OF REPLICATIONS: Test concentrations were performed in triplicate

EVALUATION PROCEDURE: Following the total incubation period, revertants were scored with an automatic counter.

OTHER: The sterility of the test solution was checked during the preliminary test. The sterility of the S9 mix was checked during each of the mutagenicity tests at the beginning and end of the experiment.
Evaluation criteria:
The study was considered valid if the following were met:
- The number of revertants of the controls was within the range of historical control data
- The number of revertants of the positive controls was higher than that of the controls and within the range of historical data

Biological relevance of the results was considered first. In addition, the following criteria were used as an aid in the determination of a positive result:
- a dose-related increase in the number of revertants and/or
- a reproducible increase in the number of revertants (doubling in at least one strain when compared to that of the controls) for at least one of the doses.

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:
yes
Remarks:
(> 2000 µg/plate)
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(> 2000 µg/plate)
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
- Preliminary Toxicity Study
The test material was freely soluble in the vehicle at 50 mg/mL in active material. Consequently, with a maximum dose volume of 100 μL/plate, the doses were 10, 100, 500, 1000, 2500 and 5000 μg/plate expressed in active material.
No precipitation, but slight to strong yellow colouration was observed in the Petri plate when scoring the revertants from 500 to 5000 μg/plate, renders scoring impossible at 2500 and 5000 μg/plate.
No toxicity was noted towards the strains with or without S9 mix.
The sterility of the test material was found to be satisfactory.

- Mutagenicity Study
The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria and were within the range of historical data for the test lab.
The top dose was selected as concentrations higher would interfere with scoring the plates. Slight toxicity was noted towards some of the strains as shown by a decrease in the number of revertants.
The test material did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the tester strains used in the study.
In both mutagenicity tests colouration of the agar was noted from a test concentrations of 144.5 µg/plate increasing in intensity up to the top dose of 2000 µg/plate.
No precipitation was recorded.
No signs of toxicity were recorded.
The sterility of the S9 mix was found to be satisfactory.

Any other information on results incl. tables

Table 1: First Mutagenicity Test

Strain

Dose

Without S9

With S9

Mean revertants/ plate

Ratio

Mean revertants/ plate

Ratio

TA 1535

0

19

-

17

-

125

14

0.8

13

0.8

250

15

0.8

17

1.0

500

16

0.8

17

1.0

1000

15

0.8

13

0.8

2000

15

0.8

13

0.8

NaN3 / 2AM

502

26.9

229

13.8

TA 1537

0

9

-

15

-

125

7

0.7

6

0.4

250

6

0.6

8

0.5

500

5

0.5

8

0.6

1000

4

0.5

6

0.4

2000

4

0.5

8

0.5

9AA / 2AM

300

32.2

246

16.4

TA 98

0

19

-

25

-

114.5

14

0.7

11

0.4

229

11

0.6

7

0.3

458

12

0.6

9

0.4

916

12

0.6

12

0.5

1832

14

0.7

6

0.2

2NF / CR

142

7.3

270

10.6

TA 100

0

95

99

125

86

0.9

78

0.8

250

94

1.0

99

1.0

500

93

1.0

87

0.9

1000

87

0.9

76

0.8

2000

82

0.9

75

0.8

NaN3 / 2AM

541

5.7

1552

15.7

TA 102

0

259

-

305

-

125

181

0.7

284

0.9

250

256

1.0

285

0.9

500

173

0.7

250

0.8

1000

268

1.0

222

0.7

2000

186

0.7

217

0.7

MMC / 2AM

2528

9.7

2204

7.2

Table 2: Second Mutagenicity Test

Strain

Dose

Without S9

With S9

Mean revertants/ plate

Ratio

Mean revertants/ plate

Ratio

TA 1535

0

15

-

19

-

125

11

0.8

14

0.7

250

8

0.6

16

0.9

500

14

0.9

18

0.9

1000

11

0.8

13

0.7

2000

12

0.8

13

0.7

NaN3 / 2AM

970

64.7

964

50.8

TA 1537

0

9

-

12

-

125

6

0.7

9

0.7

250

7

0.8

10

0.9

500

7

0.8

7

0.6

1000

9

1.1

5

0.4

2000

5

0.5

4

0.3

9AA / 2AM

1879

216.8

94

8.1

TA 98

0

23

-

29

-

125

18

0.8

18

0.6

250

14

0.6

16

0.5

500

15

0.7

18

0.6

1000

12

0.5

25

0.9

2000

10

0.5

8

0.3

2NF / CR

157

6.9

157

5.4

TA 100

0

75

-

117

-

125

66

0.9

87

0.7

250

63

0.8

71

0.6

500

70

0.9

62

0.5

1000

61

0.8

89

0.8

2000

51

0.7

76

0.7

NaN3 / 2AM

740

9.8

949

8.1

TA 102

0

269

-

391

-

125

243

0.9

433

1.1

250

275

1.0

293

0.7

500

288

1.1

141

0.4

1000

283

1.0

183

0.5

2000

220

0.8

111

0.3

MMC / 2AM

1641

6.1

879

2.2

Applicant's summary and conclusion

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

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA 102 both in the presence and absence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471 and EU Method B.14. Five strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were treated in the presence and absence of a metabolic activation system (S9 mix). In two separate assays, the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used in the presence or absence of metabolic activation.