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Toxicological information

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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
24 - 27 Jan 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions analytical purity of the test substance not specified).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
analytical purity of test substance not specified;
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Five dose levels from 50 to 5000 µg/plate (50, 150, 500, 1500 and 5000 µg/plate) in two separate experiments.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N -etþl-N'-nitro -N-nitroso guanidine (ENNG, 3µg/plate (TA100), 5µg/plate (TA1535)); 9-Aminoacridine (9AA, 80µg/plate (TA1537)); Mitomycin C (MMC, 0.5µg/plate (TA102); 4-Nitroquinoline- 1 -oxide (4NQO, 0.2 µg/plate (TA98))
Remarks:
without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2-AA, 1µg/plate, TA 100; 2µg/plate, TA 1535 and TA 1537); 1,8 Dihydroxyanthraquinone (DAN, 10µg/plate, TA 102); Benzo(a)pyren (BP, 5µg/plate, TA 98)
Remarks:
with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 37 °C for approximately 48 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: Reduction of growth of bacterial background lawn

The frequency of revertant colonies was assessed using a Domino colony counter.
Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls compared to historical controls.
- The appropriate characteristics for each tester strain have been confirmed.
- All tester strain cultures should be in the approximate range of bacteria per ml.
- Each mean positive control value should be at least two times the respective vehicle control value for each strain.
- There should be a minimum of four non-toxic test material dose levels and no evidence of excessive contamination.

The test material may be considered positive in this test system if the following criteria are met:
- The test material should have induced a reproducible, dose-related and statistically signifrcant increase in the revertant count in at least one strain of
bacteria.
Statistics:
Mean and standard deviation were calculated. Significance was tested using Dunnett's method of linear regression (Kirkland DJ (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press.)

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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.
The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.
A globular, oily, opaque precipitate was observed at 5000 mg/plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
The test material was non-toxic to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
A history profile of vehicle and positive control values was presented.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a preliminary toxicity study the test material was non-toxic to the Salmonella strain TA 100.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results from Experiment 1:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

TA 100

TA1535

TA102

TA98

TA1537

-

0

90

20

317

23

18

-

50

94

19

333

22

17

-

150

96

20

315

21

14

-

500

96

24

343

23

19

-

1500

94

26

357

27

20

-

5000

101

24

349

22

15

Positive

controls

- S9

Name

ENNG

ENNG

MMC

4NQO

9AA

Concentrations

(μg/plate)

3.0

5.0

0.5

02

80

Number of colonies/plate

439

464

740

96

1510

+

0

98

16

380

37

25

+

50

102

15

375

42

25

+

150

101

15

370

40

27

+

500

110

14

355

44

17

+

1500

104

17

358

44

24

+

5000

103

15

358

38

30

Positive

controls

+ S9

Name

2AA

2AA

DAN

BP

2AA

Concentrations

(μg/plate)

1.0

2.0

10

5

2.0

Number of colonies/plate

2112

269

06

232

217

 

Table 2: Results from Experiment 2:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

TA 100

TA1535

TA102

TA98

TA1537

-

0

76

21

274

26

11

-

50

68

23

270

25

8

-

150

81

24

300

21

9

-

500

80

25

281

23

10

-

1500

85

25

290

24

12

-

5000

76

26

280

24

14

Positive

controls

- S9

Name

ENNG

ENNG

MMC

4NQO

9AA

Concentrations

(μg/plate)

3.0

5.0

0.5

02

80

Number of colonies/plate

679

933

1167

188

2357

+

0

78

14

373

39

19

+

50

77

14

385

46

27

+

150

86

15

401

45

21

+

500

82

14

382

42

19

+

1500

85

16

400

44

20

+

5000

91

19

379

39

16

Positive

controls

+ S9

Name

2AA

2AA

DAN

BP

2AA

Concentrations

(μg/plate)

1.0

2.0

10

5

2.0

Number of colonies/plate

3159

313

1021

312

222

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

DAN: 1,8 Dihydroxyanthraquinone

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

MMC: Mitomycin C

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative Under the tested experimental conditions the test substance did not induce gene mutations in S. typhimurium strains up to the limit dose.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.