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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:
4 November 2004 to 10 December 2004
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

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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
Qualifier:
according to
Guideline:
other: USA EPS (TSCA) OPPTS harmonised guidelines
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
(the method conforms with those published by METI, MHLW and MAFF)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: opaque liquid
Details on test material:
- Appearance: Blue opaque liquid
- Storage conditions: Room temperature in the dark

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
10 % liver S9 in standard co-factors
Test concentrations with justification for top dose:
- Preliminary Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Mutation Test (experiments 1 and 2): 0, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water. Formulated concentrations were adjusted to make allowances for the purity of the test material.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene, 1,8-dihydroxyanthraquinone
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);
A measured aliquot (0.1 mL) of bacterial culture was dispensed into a tube followed by 2.0 mL of molten, trace histidine-supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9 mix or phosphate buffer. The content of the tube was dispensed onto the surface of a Vogel-Bonner minimal agar plate and incubated.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The procedure was repeated in triplicate for each concentration in each bacterial strain for both Experiment 1 and Experiment 2.

NUMBER OF CELLS EVALUATED: The frequency of revertant colonies was assessed using a Domino colony counter. The top dose level was assessed manually due to test material-induced colouration.

DETERMINATION OF CYTOTOXICITY
- Method: Assessment of effects on the growth of the background bacterial lawn.
Evaluation criteria:
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 significant increase in the revertant count of at least one strain of bacteria.
Statistics:
Dunnett’s method of linear regression was used to assess statistical significance.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test material caused no visible reduction in the growth of the background bacterial lawn at any dose level. A blue colour was observed at and above 50 µg/plate, becoming darker with increasing concentration. This did not prevent scoring of revertant colonies. No test material precipitate was observed on any of the plates at any of the dose levels tested in either the presence or the absence of S9 mix.
No significant increases in the frequency of revertant colonies were recorded for any of the strains at any dose level with or without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1 Summary of Results

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

TA102

TA98

TA1537

-

-

-

-

-

-

0

50

150

500

1500

5000

132

148

140

144

146

132

31

30

32

29

20

19

304

339

350

303

353

338

23

20

27

25

16

20

15

18

12

16

13

12

+

+

+

+

+

+

0

50

150

500

1500

5000

134

120

138

132

105

125

16

10

10

11

12

9

374

377

345

283

376

374

37

42

44

41

28

28

19

17

20

19

17

18

                                                     Positive Controls

-

Name

ENNG

ENNG

MMC

4NQO

9AA

Concentration (µg/plate)

3

5

0.5

0.2

80

Mean no. colonies/plate

740

621

1213

164

629

+

Name

2AA

2AA

DAN

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

Mean no. colonies/plate

802

231

1029

245

437

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

MMC = mitomycin C

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

DAN = 1,8-dihydroxyanthraquinone

 

Table 2: Experiment 2 Summary of Results

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

TA102

TA98

TA1537

-

-

-

-

-

-

0

50

150

500

1500

5000

88

78

86

77

90

72

20

19

14

17

19

15

371

348

364

341

258

335

17

15

12

17

18

12

14

20

15

12

10

13

+

+

+

+

+

+

0

50

150

500

1500

5000

79

87

92

93

85

78

11

12

11

11

10

12

352

354

349

348

343

360

28

37

36

29

34

20

19

15

23

16

14

17

                                                     Positive Controls

-

Name

ENNG

ENNG

MMC

4NQO

9AA

Concentration (µg/plate)

3

5

0.5

0.2

80

Mean no. colonies/plate

338

465

1108

135

490

+

Name

2AA

2AA

DAN

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

Mean no. colonies/plate

566

233

905

254

376

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

MMC = mitomycin C

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

DAN = 1,8-dihydroxyanthraquinone

Applicant's summary and conclusion

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

Under the conditions of this study, the test material was found to be devoid of mutagenic activity on Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA 100 and TA 102 in the presence and absence of metabolic activation.
Executive summary:

The mutagenic potential of the test material was investigated in a bacterial reverse mutation assay which was conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14, USA EPS (TSCA) OPPTS harmonised guidelines and those published in Japan by METI, MHLW and MAFF under GLP conditions.

Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA100 and TA 102 were treated with the test material (using the Ames plate incorporation method) at dose levels of 50, 150, 500, 1500 and 5000 µg/plate in distilled water both in the presence and absence of metabolic activation (S9-mix). Concurrent solvent, negative and positive controls were run.

The tets material caused no visible reduction in the growth of the background bacterial lawn at any dose level. A blue colour was observed at and above 50 µg/plate, becoming darker with increasing concentration. This did not prevent scoring of revertant colonies. No test material precipitate was observed on any of the plates at any of the dose levels tested in either the presence or the absence of S9 mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains at any dose level with or without metabolic activation.

Under the conditions of this study, the test material was found to be devoid of mutagenic activity on Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA 100 and TA 102 in the presence and absence of metabolic activation.