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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 from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
22 Nov - 21 Dec 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (no test substance purity specified).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

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
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Physical state: transparent colorless liquid
- Analytical purity: not specified
- Lot/batch No.: 7054429
- Storage condition of test material: at room temperature

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment I:
Salmonella strains: 10, 33.3, 100, 333, 1000, 5000 µg/plate (with and without S9 mix)
E.coli strain: 33.3, 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix)
Experiment II:
all strains: 33.3, 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
other: 2-aminoanthracene (2-AA, all, 2.5 + 5.0 µg/plate, +S9); ICR-191 (TA1537, 2.0 µg/plate, -S9); sodium azide (SA, TA100 + 1535, 2.0 µg/plate, -S9); 2-nitrofluorene (2NF, TA98, 1.0 µg/plate, -S9); 4-nitroquinoline-N-oxide (4NQO, WP2, 2.0 µg/plate, -S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: density of bacterial background lawn

Evaluation criteria:
1) Tester strains TA98, TA100 and WP2 uvrA
For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
2) Tester strain TA1535 and TA1537
For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least on of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose-response to increasing concentrations of the test article.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed

RANGE-FINDING/SCREENING STUDIES:
The cytotoxicity of the test item was determined in TA100 and WP2 uvrA in order to allow selection of appropriate doses to be tested in the mutagenicity assay. The test article was tested up to a maximum concentration of 5 mg/plate (dose range 6.67 - 5000 µg/plate). Cytotoxicity was observed with tester strain TA100 at 667 µg/plate and above in the presence of S9 mix and 333 µg/plate and above in the absence of S9 mix as evidenced by thinning of the bacterial background lawn. No cytotoxicity was observed for tester strain WP2 uvrA. In general no test article precipitate was observed. The highest dose chosen for the main study gave a reduction of revertants per plate and/or thinning or disappearance of the bacterial background lawn.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study no changes of the background lawn were observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. 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

TA 100

TA1535

WP2 uvrA

TA98

TA1537

vehicle

97 ± 6

11 ± 4

184 ± 28

23 ± 4

9 ± 3

10

101 ± 1

13 ± 3

--

18 ± 3

5 ± 1

33.3

100 ± 8

14 ± 5

175 ± 6

16 ± 3

5 ± 3

100

117 ± 12

13 ± 2

191 ± 8

20 ± 4

3 ± 1

333

94 ± 5

12 ± 2

183 ± 10

19 ± 6

6 ± 3

1000

101 ± 10

9 ± 1

197 ± 16

17 ± 6

5 ± 2

3330

--

--

161 ± 17

--

--

5000

100 ± 21

14 ± 4

188 ± 7

22 ± 3

6 ± 3

Positive controls, –S9

Name

SA

SA

4NQO

2NF

ICR-191

Concentrations

(μg/plate)

2

2

2

1

2

Mean No. of colonies/plate

(average of 3 ± SD)

751 ± 35

590 ± 44

1319 ± 59

298 ± 19

427 ± 36

+

vehicle

98 ± 3

13 3

197 ± 39

30 ± 5

4 ± 2

+

10

130 ± 10

16 3

--

33 ± 4

7 ± 2

+

33.3

133 ± 6

12 5

186 ± 12

34 ± 5

8 ± 0

+

100

124 ± 5

13 4

198 ± 20

34 ± 4

7 ± 0

+

333

143 ± 8

16 2

203 ± 9

31 ± 2

6 ± 1

+

1000

138 ± 8

15 5

226 ± 64

30 ± 2

9 ± 1

+

3330

--

--

178 ± 12

--

--

+

5000

130 ± 8

14 3

204 ± 15

25 ± 3

4 ± 2

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

5

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

774 ± 19

134 ± 6

840 ± 191

804 ± 36

128 ± 9

 

4NQO = 4-nitroquinoline-N-oxide

2AA = 2-Aminoanthracene

SA = Sodium Azide

2NF = 2-Nitrofluorene

 

Table 2. 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

TA 100

TA1535

WP2 uvrA

TA98

TA1537

vehicle

96 ± 17

17 ± 3

159 ± 28

12 ± 1

6 ± 3

33.3

99 ± 7

15 ± 5

135 ± 10

15 ± 4

6 ± 2

100

90 ± 4

20 ± 5

142 ± 12

12 ± 3

4 ± 1

333

103 ± 9

15 ± 6

120 ± 19

14 ± 4

4 ± 1

1000

109 ± 11

13 ± 5

138 ± 27

19 ± 3

8 ± 1

3330

108 ± 10

11 ± 5

150 ± 19

17 ± 3

5 ± 1

5000

95 ± 11

13 ± 4

150 23

15 ± 2

5 ± 2

Positive controls, –S9

Name

SA

SA

4NQO

2NF

ICR-191

Concentrations

(μg/plate)

2

2

2

1

2

Mean No. of colonies/plate

(average of 3 ± SD)

581 ± 33

523 ± 13

1355 ± 87

268 ± 42

411 ± 64

+

vehicle

85 ± 9

16 ± 5

178 ± 18

18 ± 4

6 ± 1

+

33.3

93 ± 25

10 ± 1

131 ± 14

15 ± 4

7 ± 2

+

100

97 ± 18

11 ± 2

141 ± 19

17 ± 3

7 ± 2

+

333

88 ± 10

14 ± 5

139 ± 6

24 ± 6

7 ± 1

+

1000

104 ± 15

12 ± 1

156 ± 14

21 ± 3

9 ± 4

+

3330

91 ± 15

8 ± 3

138 ± 6

27 ± 3

9 ± 2

+

5000

89 ± 8

8 ± 3

127 ± 6

24 ± 10

8 ± 2

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

5

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

834 ± 16

105 ± 17

1019 ± 50

842 ± 65

127 ± 11

 

4NQO = 4-nitroquinoline-N-oxide

2AA = 2-Aminoanthracene

SA = Sodium Azide

2NF = 2-Nitrofluorene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results of the assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of metabolic activation.