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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:
February 9th, 2009 - February 23rd, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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
Details on test material:
- Name of test material (as cited in study report): SH-1
- Chemical name of test material (as cited in study report): Benzene, 1,1´-(1,2-ethanediyl)bis-, brominated
- Physical state: solid
- Analytical purity: > 99 %
- Batch No.: 20081010
- Expiration date of the lot/batch: January 08, 2011.
- Storage condition of test material: at room temperature

Method

Target gene:
Genes involved in Histidine synthesis
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment = Experiment I (plate incorporation test):
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II (pre-incubation test):
33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 4-nitro-o-phenylene-diamine, methyl methane sulfonate, 2-aminoanthracene,
Details on test system and conditions:
METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation);
Experiment II: preincubation;

Experimental Performance:

The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar

In the pre-incubation assay 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After preincubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Statistics:
no statistics performed, as this is not mandatory according to OECD guideline 471.

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:
no, but tested up to limit concentrations
Remarks:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was observed in the test tubes from 1000 - 5000 µg/plate in experiment I, and at 2500 and 5000 µg/plate in experiment II. Precipitation of the test item was also observed on the incubated agar plates at 2500 and 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I.
Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades.

COMPARISON WITH HISTORICAL CONTROL DATA: the negative controls were within the ranges of the historical control values.

Any other information on results incl. tables

Table 1: Ames Test Results of the plate incorporation Experiment I (= Pre-Experiment):

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

-

Neg. control

21

8

28

144

55

-

0

15

11

29

142

55

-

3

20

12

26

136

49

-

10

15

10

25

128

54

-

33

18

8

28

138

52

-

100

19

10

23

147

49

-

333

20

8

24

143

58

-

1000

18

10

29

134

59

-

2500

19P

11P

28P

128P

49P

-

5000

16P

8P

19P

121P

50P

Positive

controls

- S9

Name

NaN3

4-NOPD

4-NOPD

NaN3

MMS

Concentration

(per plate)

10 μg

50 μg

10 μg

10 μg

3.0 μL

Number of colonies/plate

1966

92

439

2019

1390

+

Neg. control

15

12

32

155

63

+

0

16

11

28

161

62

+

3

17

13

32

163

62

+

10

17

9

30

157

61

+

33

19

11

28

153

61

+

100

18

14

26

154

63

+

333

19

11

30

167

57

+

1000

21

13

31

155

58

+

2500

20P

13P

27P

140P

58P

+

5000

17P

7P

22P

128P

56P

Positive

controls

+ S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

(per plate)

2.5 μg

2.5 μg

2.5 μg

2.5 μg

10 μg

Number of colonies/plate

379

265

1859

2699

237

 

 

Table 2: Ames Test Results of the preincubation Experiment II:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

-

Neg. control

18

10

26

142

51

-

0

19

13

25

126

52

-

33

19

11

26

122

49

-

100

15

12

26

120

48

-

333

18

8

24

117

48

-

1000

12

10

22

121

49

-

2500

15P

12P

27P

121P

56P

-

5000

11P

9P

22P

111P

46P

Positive

controls

- S9

Name

NaN3

4-NOPD

4-NOPD

NaN3

MMS

Concentration

per plate

10 μg

50 μg

10 μg

10 μg

3.0 μL

Number of colonies/plate

2008

91

415

2037

353

+

Neg. control

18

13

30

170

49

+

0

19

10

37

152

56

+

33

16

12

37

144

53

+

100

17

9

36

157

56

+

333

16

12

33

151

58

+

1000

15

10

38

145

57

+

2500

18P

11P

33P

133P

55P

+

5000

14P

12P

26P

151P

48P

Positive

controls

+ S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

per plate

2.5 μg

2.5 μg

2.5 μg

2.5 μg

10 μg

Number of colonies/plate

304

204

1691

2358

203

 

NaN3 = Sodium azide

MMS = Methyl methane sulfonate

4-NOPD = 4-nitro-o-phenylene-diamine

2-AA = 2-aminoanthracene

P = Precipitate

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, the test substance SH-1 was shown to be non-mutagenic in bacteria.
Executive summary:

This study was performed to investigate the potential of SH-1 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the

Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with SH-1 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, SH-1 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.