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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:
experimental study
Adequacy of study:
key study
Study period:
21 Dec 2009 - 01 Feb 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Target gene:
his operon (S. typhimurium strains)
trp operon (E. coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 80 mg/kg bw/day phenobarbitone and 100 mg/kg bw/day beta-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1 and 2: 50, 150, 500, 1500 and 5000 μg/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble in DMSO at 50 mg/mL
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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
other: 2-aminoanthracene
Remarks:
See 'Details on test system and conditions' for concentrations and strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, experiment 1) and preincubation (experiment 2)

DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replication each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in growth of bacterial background lawn

OTHER:
- positive control substances used: N-ethyl-N'-nitro-N-nitrosoguanidine (2 μg/plate in DMSO, -S9, WP 2urvA-; 3 μg/plate, -S9, TA 100; 5 μg/plate, -S9, TA 1535); 9-aminoacridine (80 μg/plate in DMSO, -S9, TA 1537); 4-nitroquinoline-1-oxide (0.2 μg/plate in DMSO, -S9, TA 98); 2-aminoanthracene (1 μg/plate in DMSO, +S9, TA 100; 2 μg/plate, +S9 TA 1535 and TA 1537; 10 μg/plate, +S9, WP 2urvA-); benzo-a-pyrene (5 μg/plate in DMSO, +S9, TA 98)
Evaluation criteria:
There are several criteria for determining a positive result, such as the 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. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Results and discussion

Test resultsopen allclose all
Key result
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
Remarks:
precipitation was observed from 1500 μg/plate in experiment 1 and from 500 μg/plate in experiment 2, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed from 1500 μg/plate in experiment 1 and from 500 μg/plate in experiment 2, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitate was observed at concentrations at and above 1500 μg/plate in experiment 1 and concentrations at and above 500 μg/plate in experiment 2, with and without metabolic activation, respectively. This did not affect the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: A toxicity screening study was performed to determine the toxicity of the test substance. 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate test substance was tested on TA 100 and E. coli WP2 uvrA using the plate incorporation method. Precipitation of the test material was observed at 5000 μg/plate. A range-finding study (plate incorporation method) was performed on all strains using dose levels of 50 - 5000 μg/plate. As the results for all the tested dose levels were valid, the results were used as part of the main study (experiment 1).

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the results of the vehicle and positive controls fell within the range of the historical control data (see Table 3 under 'Any other information on results incl. tables')

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed at any dose level.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation method)

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

TA 100

TA1535

WP2 uvR

TA98

TA1537

DMSO

134 ± 5.7

27 ± 2.3

28 ± 0.6

26 ± 2.0

11 ± 5.0

50

136 ± 4.4

25 ± 0.0

26 ± 2.5

27 ± 0.6

14 ± 1.5

150

131 ± 6.4

29 ± 3.8

25 ± 6.7

24 ± 4.5

11 ± 3.8

500

115 ± 21.7

28 ± 1.5

27 ± 1.2

25 ± 1.0

17 ± 1.0

1500*

106 ± 18.0

25 ± 4.2

22 ± 4.7

22 ± 3.6

10 ± 2.6

5000*

116 ± 5.1

32 ± 2.1

16 ± 1.7

19 ± 1.2

10 ± 2.1

Positive controls,

-S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

429 ± 43.7

514 ± 43.1

726 ± 77.5

133 ± 11.0

884 ± 92.2

+

DMSO

102 ± 11.6

12 ± 2.5

25 ± 3.5

28 ± 2.3

12 ± 3.2

+

50

109 ± 10.4

12 ± 1.5

27 ± 0.6

28 ± 3.5

15 ± 1.0

+

150

106 ± 3.2

12 ± 1.0

29 ± 0.6

28 ± 1.2

11 ± 4.0

+

500

97 ± 1.5

14 ± 1.7

26 ± 5.0

25 ± 2.1

13 ± 2.6

+

1500*

101 ± 8.1

14 ± 1.2

24 ± 3.5

26 ± 2.6

11 ± 2.0

+

5000*

93 ± 3.0

13 ± 2.6

26 ± 2.5

27 ± 1.5

9 ± 2.6

Positive controls, +S9

Name

2AA

2AA

2AA

BaP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

1547 ± 34.6

223 ± 24.5

367 ± 41.1

218 ± 74.1

299 ± 29.5

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

4NQO: 4-nitroquinoline N-oxide

9AA:9-aminocridine

2AA: 2-amino-anthracene

BaP: benzo-a-pyrene

*: precipitation

 

Table 2. Test results of experiment 2 (plate incorporation method)

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

TA 100

TA1535

WP2 uvR

TA98

TA1537

DMSO

102 ± 12.7

24 ± 2.5

22 ± 6.1

18 ± 4.4

16 ± 2.5

50

85 ± 12.4

24 ± 5.0

21 ± 2.6

15 ± 2.3

13 ± 1.2

150

94 ± 7.1

24 ± 2.9

18 ± 2.5

17 ± 5.1

13 ± 1.7

500*

90 ± 10.0

23 ± 4.0

21 ± 0.6

20 ± 2.1

15 ± 6.1

1500*

94 ± 5.1

26 ± 3.5

22 ± 1.5

20 ± 2.6

14 ± 1.5

5000*

96 ± 4.5

24 ± 2.3

23 ± 3.5

20 ± 2.0

14 ± 2.0

Positive controls,

-S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

613 ± 32.2

578 ± 58.0

861 ± 68.6

144 ± 9.5

354 ± 120.8

+

DMSO

99 ± 8.5

16 ± 2.1

28 ± 5.1

27 ± 6.7

14 ± 3.2

+

50

90 ± 4.2

15 ± 1.0

29 ± 1.5

25 ± 4.0

10 ± 1.5

+

150

94 ± 6.7

14 ± 1.5

25 ± 3.6

25 ± 8.2

14 ± 1.5

+

500*

98 ± 12.7

15 ± 1.2

24 ± 3.1

28 ± 1.7

14 ± 2.0

+

1500*

98 ± 2.1

16 ± 3.0

25 ± 3.6

26 ± 4.4

16 ± 2.5

+

5000*

97 ± 4.7

12 ± 1.5

26 ± 1.5

25 ± 2.3

12 ± 1.5

Positive controls, +S9

Name

2AA

2AA

2AA

BaP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

586 ± 96.2

235 ± 12.0

259 ± 36.7

387 ± 51.7

165 ± 8.4

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

4NQO: 4-nitroquinoline N-oxide

9AA:9-aminocridine

2AA: 2-amino-anthracene

BaP: benzo-a-pyrene

*: precipitation

Table 3. Historical controls

Strain

Control

without S9-mix

with S9-mix

Year

 

 

Mean ± SD

Mean ± SD

 

TA100

Solvent/untreated

94 ± 16.0

87 ± 14.8

2007

TA100

Positive

558 ± 313.5

1231 ± 651.1

2007

TA1535

Solvent/untreated

21 ± 5.2

13 ± 3.6

2007

TA1535

Positive

343 ± 864.2

231 ± 476.5

2007

WP2 uvR

Solvent/untreated

26 ± 5.8

28 ± 6.4

2007

WP2 uvR

Positive

652 ± 707.9

505 ± 535.2

2007

TA98

Solvent/untreated

21 ± 5.5

26 ± 6.0

2007

TA98

Positive

256 ± 289.7

308 ± 280.5

2007

TA1537

Solvent/untreated

11 ± 3.8

13 ± 4.1

2007

TA1537

Positive

1697 ± 1193.6

339 ± 254.4

2007

TA100

Solvent/untreated

105 ± 18.7

101 ± 18.5

2008

TA100

Positive

487 ± 184.7

1138 ± 517.2

2008

TA1535

Solvent/untreated

23 ± 5.3

15 ± 4.8

2008

TA1535

Positive

210 ± 107.7

272 ± 94.9

2008

WP2 uvR

Solvent/untreated

26 ± 5.9

29 ± 7.2

2008

WP2 uvR

Positive

548 ± 231.5

426 ± 273.9

2008

TA98

Solvent/untreated

19 ± 4.6

26 ± 5.3

2008

TA98

Positive

155 ± 70.8

247 ± 178.2

2008

TA1537

Solvent/untreated

13 ± 3.7

13 ± 3.8

2008

TA1537

Positive

930 ± 476.2

279 ± 101.7

2008

 

Applicant's summary and conclusion