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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Sep - 9 Oct 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
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
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone (80/100 mg/kg bw/day for 3 consecutive days)
Test concentrations with justification for top dose:
Pre-experiment:
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

First and second experiment: 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate with and without metabolic activation
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was fully soluble at 50 mg/mL in DMSO.
Controls
Negative controls:
yes
Remarks:
untreated controls
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-dihydroxyanthraquinone (DAN), 2-aminoanthracene (2-AA), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG), 9-aminoacridine (9-AA), mitomycin C (MMC), 4-nitroquinoline-N-oxide (4-NQO), benzo(a)pyrene (BaP)
Remarks:
+S9: 2-AA (1 µg/plate, TA100; 2 µg/plate, TA1535, TA1537); BaP (5 µg/plate, TA98); DAN (10 µg/plate, TA102) -S9: ENNG (3 µg/plate, TA100; 5 µg/plate, TA1535); 9-AA (80 µg/plate, TA1537); MMC (0.5 µg/plate, TA102); 4-NQO (0.2 µg/plate, TA98)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony frequency or reduction of the bacterial background lawn
Evaluation criteria:
Several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation, were considered. However, biological relevance of the results was considered first. Additionally, statistical methods were used as an aid to evaluate a positive response.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(exp. 1: -S9: at 150 µg/plate in TA1535 and TA102; at 500 µg/plate in remaining strains; +S9: at 150 µg/plate in TA102; at 500 µg/plate in rem. strains; exp. 2: -S9: at 150 µg/plate; +S9: at 500 µg/plate in TA1537; at 150 µg/plate in rem. strains)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for the main study, a preliminary experiment was carried out to determine the toxicity of the test material. The test substance was toxic at and above 1500 µg/plate in TA100.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance caused a visible reduction in the growth of the bacterial background lawn of all tester strains in both the presence and absence of metabolic activation, initially from 150 µg/plate for TA100 and 500 µg/plate for the remaining tester strains. Several of the bacterial strains also exhibited substantial decreases in revertant colony frequency at 150 µg/plate in both the presence and absence of metabolic activation. The toxicity of the test substance to the tester strains varied slightly between experiment number and strain type and was of sufficient severity to prevent the test substance from being tested up to the maximum recommended dose level of 5000 µg/plate.

Any other information on results incl. tables

Table 2. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

135

25

316

14

9

0 (DMSO)

138 ± 21.7

28 ± 2.3

350 ± 4.9

14 ± 0.6

10 ± 0.6

1.5

145 ± 19.7

30 ± 5.0

329 ± 19.1

16 ± 5.7

9 ± 2.0

5

149 ± 8.7

31 ± 6.7

349 ± 50.1

18 ± 6.8

9 ± 0.6

15

134 ± 20.5

25 ± 4.6

360 ± 10.8

12 ± 3.8

10 ± 2.5

50

124 ± 8.5

27 ± 5.5

278 ± 34.9

17 ± 4.4

9 ± 1.2

150

151 ± 3.2

11 ± 1.0

91 ± 14.2

14 ± 0

6 ± 1.2

500

0T

0T

0T

0T

0T

1500

0T

0T

0T

0T

0T

Positive controls, –S9

Name

ENNG

ENNG

MMC

4-NQO

9-AA

Concentrations

[μg/plate]

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

495 ± 25.2

319 ± 19.1

2525 ± 5.5

455 ± 22.7

626 ± 48.2

+

0 (DMSO)

146 ± 25.1

17 ± 7.0

369 ± 16.2

24 ± 5.2

11 ± 3.6

+

1.5

143 ± 14.2

14 ± 8.4

385 ± 12.5

26 ± 1.5

12 ± 1.0

+

5

140 ± 20.0

11 ± 2.1

364 ± 28.9

25 ± 2.0

12 ± 2.1

+

15

129 ± 7.9

14 ± 7.0

311 ± 11.0

21 ± 1.2

13 ± 4.4

+

50

116 ± 13.4

14 ± 1.2

320 ± 12.1

19 ± 1.2

12 ± 6.2

+

150

96 ± 11.5

11 ± 2.3

188 ± 14.0

15 ± 5.2

7 ± 2.1

+

500

0T

0V

0T

0T

0T

+

1500

0T

0T

0T

0T

0T

Positive controls, +S9

Name

2-AA

2-AA

DAN

BaP

2-AA

Concentrations

[μg/plate]

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

660 ± 49.0

498 ± 53.6

1080 ± 95.2

435 ± 80.0

397 ± 33.3

DMSO: dimethylsulphoxide

BaP: benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

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

4-NQO: 4-nitroquinoline-N-oxide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

MMC: mytomycin C

T: toxic, no bacterial background lawn

V: very weak bacterial background lawn

 

Table 3. Test results of Experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

101

20

318

19

11

0 (DMSO)

94 ± 9.7

30 ± 1.5

275 ± 13.3

19 ± 3.0

14 ± 2.6

1.5

88 ± 4.6

30 ± 2.3

256 ± 15.8

16 ± 4.4

10 ± 1.5

5

95 ± 11.9

31 ± 7.8

261 ± 5.7

15 ± 6.0

11 ± 1.0

15

87 ± 5.5

26 ± 2.9

230 ± 20.1

16 ± 1.5

13 ± 2.1

50

96 ± 16.1

20 ± 2.1

229 ± 18.6

13 ± 8.5

13 ± 3.1

150

32 ± 3.2S

8 ± 1.2

45 ± 7.6

5 ± 1.0

4 ± 0.6

500

0T

0T

0T

0T

0T

1500

0T

0T

0T

0T

0T

Positive controls, –S9

Name

ENNG

ENNG

MMC

4-NQO

9-AA

Concentrations

[μg/plate]

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

505 ± 28.2

182 ± 27.2

1541 ± 68.8

131 ± 2.5

884 ± 243.8

+

0 (DMSO)

86 ± 4.0

12 ± 1.7

322 ± 9.9

21 ± 2.6

12 ± 3.8

+

1.5

85 ± 14.0

12 ± 4.0

290 ± 4.6

24 ± 6.0

8 ± 5.0

+

5

97 ± 9.2

14 ± 2.5

251 ± 20.0

24 ± 1.2

15 ± 3.5

+

15

86 ± 9.9

13 ± 4.2

247 ± 20.3

16 ± 2.6

10 ± 4.6

+

50

76 ± 5.3

11 ± 0.6

216 ± 20.0

20 ± 2.3

9 ± 3.5

+

150

34 ± 7.2S

5 ± 1.5

111 ± 5.3

5 ± 0.6

7 ± 3.6

+

500

0T

0T

0T

0T

0T

+

1500

0T

0T

0T

0T

0T

Positive controls, +S9

Name

2-AA

2-AA

DAN

BaP

2-AA

Concentrations

[μg/plate]

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

596 ± 45.5

275 ± 42.0

1407 ± 114.5

203 ± 16.8

183 ± 37.5

DMSO: dimethylsulphoxide

BaP: benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

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

4-NQO: 4-nitroquinoline-N-oxide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

MMC: mytomycin C

S: sparse bacterial background lawn

T: toxic, no bacterial background lawn

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA1535, TA1537, TA98, TA100 and TA102) tested with and without metabolic activation.
Executive summary:

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 1.5 to 1500 µg/plate in the first experiment. The experiment was repeated using the same dose range as Experiment 1. Additional dose levels and an expanded dose range were selected to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn of all tester strains in both the presence and absence of S9, initially from 150 µg/plate for TA100 and 500 µg/plate for the remaining tester strains. Several of the bacterial strains also exhibited substantial decreases in revertant colony frequency at 150 µg/plate in both the presence and

absence of S9. The toxicity of the test material to the tester strains varied slightly between experiment number and strain type and was of sufficient severity to prevent the test material from being tested up to the maximum recommended dose level of 5000 lag/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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.

In conclusion, the test material was considered to be non-mutagenic under the conditions of this test.