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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 based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
09 Oct - 23 Oct 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany
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
- Substance type: organic
- Physical state: amber liquid
- Analytical purity: 99% a.i.
- Lot/batch No.: ES665M0001
- Expiration date of the lot/batch: June 30, 2007
- Storage condition of test material: room temperature

Method

Target gene:
his operon
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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital i.p. and ß-Naphthoflavone p.o.
Test concentrations with justification for top dose:
first experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation
second experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen because of it´s solubility properties and its relative non-toxicity to the bacteria
Controls
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls were performed.
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent vehicle controls were performed.
True negative controls:
no
Positive controls:
yes
Remarks:
sodium azide (NaN3), 4-nitro-o-phenylene-diamine (4-NOPD), methyl-methane-sulfonate (MMS) and 2-aminoanthracene (2-AA)
Positive control substance:
other: without S9 mix: NaN3, 10 µg/plate in TA 1535 and TA 100; 4-NOPD, 10 µg/plate in TA 98, 50 µg/plate in TA 1537; MMS, 3.0 µL/plate in TA 102; with S9 mix: 2-AA, 2.5 µg/plate in TA 1535, TA 1537, TA 98, TA 100 and 10 µg/plate in TA 102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: three replicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative growth

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to reduced background growth some plates were counted manually.
Evaluation criteria:
A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed. A dose dependent increase was considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase is not considered biologically relevant
Statistics:
Mean values and standard deviation were calculated.

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Based on the toxic effects observed in the range finding study, eight concentrations were tested in main experiment and 5,000 µg/plate were chosen as maximal concentration.

ADDITIONAL INFORMATION ON CYTOTOXICITY: see tables 3 and 4
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Summary of results Pre-experiment/Experiment I

Metabolic activation

Test group

Dose Level (µg/plate)

Revertant Colony Counts (Mean±SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without activation

DMSO

 

23±11

13±0

36±3

146±7

520±46

Untreated

 

21±8

9±4

26±2

138±24

530±38

Test substance

3

19±0

12±7

34±13

149±11

513±48

10

20±3

7±2

33±6

153±27

508±10

33

14±2

10±3

31±6

146±18

525±12

100

25±3

14±3

37±12

130±14

500±3

333

23±6

10±5

36±3

116±15

412±12

1000

13±3

7±2

19±3

86±6

354±52

2500

9±1

1±1

16±2

47±10

250±26

5000

4±2

0±1

11±3

27±2

132±9

NaN3

10

1980±52

 

 

2043±204

 

4-NOPD

10

 

 

390±24

 

 

4-NOPD

50

 

106±17

 

 

 

MMS

3

 

 

 

 

2736±155

 

 

 

 

 

 

 

 

With activation

DMSO

 

21±6

17±6

48±6

156±26

628±69

Untreated

 

26±4

17±4

39±9

154±4

671±76

Test substance

3

24±3

18±11

43±6

173±6

584±41

10

26±2

15±7

37±1

174±17

643±33

33

24±4

10±4

41±6

158±11

677±16

100

24±1

12±3

38±7

164±1

662±31

333

26±5

12±3

35±6

176±8

570±49

1000

23±5

18±6

45±6

153±5

630±82

2500

21±6

11±3

39±10

143±13

575±110

5000

11±2

4±2

14±6

93±8

348±30

2-AA

2.5

400±29

701±76

3163±334

4283±196

 

2-AA

10

 

 

 

 

2495±221

 

 

 

 

 

 

 

 

DMSO = dimethylsulfoxide

NaN3 = sodium azide

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

MMS = methyl-methane-sulfonate

2-AA = 2 -aminoanthracene

Table 2. Summary of results Experiment II

Metabolic activation

Test group

Dose Level (µg/plate)

Revertant Colony Counts (Mean±SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without activation

DMSO

 

19±5

13±0

34±7

116±18

404±16

Untreated

 

20±11

11±2

32±6

150±2

444±10

Test substance

3

14±2

13±3

28±5

149±136

391±17

10

19±5

12±6

22±4

106±8

388±17

33

22±2

10±4

24±3

118±11

315±5

100

15±2

6±3

25±8

82±5

262±20

333

17±3

14±2

23±6

69±13

243±9

1000

8±1

1±1

14±2

39±17

83±19

2500

0±1

0±0

7±2

11±3

7±7

5000

0±0

0±0

0±0

0±0

0±0

NaN3

10

1672±35

 

 

1717±137

 

4-NOPD

10

 

 

524±14

 

 

4-NOPD

50

 

119±13

 

 

 

MMS

3

 

 

 

 

1718±205

 

 

 

 

 

 

 

 

With activation

DMSO

 

28±6

11±4

42±6

123±8

433±19

Untreated

 

37±15

17±3

43±6

153±12

549±46

Test substance

3

31±5

18±3

39±6

150±3

485±38

10

32±11

17±5

43±14

129±12

435±56

33

39±11

10±2

54±18

113±26

406±79

100

32±15

12±4

34±1

120±19

392±29

333

25±3

10±2

42±6

128±18

194±64

1000

20±3

8±5

28±3

72±6

58±10

2500

9±4

10±4

22±7

46±10

39±11

5000

7±2

0±0

12±3

15±5

14±3

2-AA

2.5

298±15

263±12

2097±6

2118±6

 

2-AA

10

 

 

 

 

1383±148

 

 

 

 

 

 

 

 

DMSO = dimethylsulfoxide

NaN3 = sodium azide

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

MMS = methyl-methane-sulfonate

2-AA = 2 -aminoanthracene

Table 3. Reduced backround growth were observed at the following plates:

Strain

Experiment I

 

 

Experiment II

 

 

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

5000

5000

1000-5000

5000

TA 1537

2500-5000

5000

1000-5000

5000

TA 98

2500sodium azide (NaN3), 4-nitro-o-phenylene-diamine (4-NOPD), methyl-methane-sulfonate (MMS) and 2-aminoanthracene (2-AA)-5000

5000

1000-5000

5000

TA 100

5000

5000

1000-5000

5000

TA 102

2500-5000

5000

1000-5000

1000-5000

Table 4. Toxic effects as a reduction in number of revertants were observed at the following plates:

Strain

Experiment I

 

 

Experiment II

 

 

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

2500-5000

-

1000-5000

2500-5000

TA 1537

2500-5000

5000

1000-5000

5000

TA 98

2500-5000

5000

1000-5000

5000

TA 100

2500-5000

-

1000-5000

2500-5000

TA 102

5000

-

1000-5000

333-5000

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any concentration 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. They showed a distinct increase in induced revertant colonies. Negative and positive control values were within the range of historical control data (see Table 5).

Table 5. These data represent the laboratory's historical control data from May 2005 until June 2006 representing approx. 200 experiments (TA 102 the historical control data are based on approx. 100 experiments).

Strain

 

without S9 mix

with S9 mix

 

Mean

SD

Min

Max

Mean

SD

Min

Max

TA 1535

Solvent Control

20.8

4.7

9

35

24.7

5.9

7

43

Negative Control

20.4

4.4

11

31

24.2

5.5

10

38

Positive Control

1422.0

464.7

781

1900

332.0

95.3

107

695

TA 1537

Solvent Control

11.2

3.7

5

28

16.2

5.0

6

36

Negative Control

11.6

4.0

4

28

17.1

5.4

7

34

Positive Control

99.8

32.5

53

425

276.8

132.6

59

746

TA 98

Solvent Control

28.1

6.1

15

49

37.9

7.4

20

57

Negative Control

30.2

6.6

16

60

39.0

7.5

18

64

Positive Control

439.0

155.2

176

1818

1839.4

898.6

407

4891

TA 100

Solvent Control

130.7

20.8

87

197

147.0

25.5

84

255

Negative Control

138.2

21.6

86

216

150.1

24.2

96

214

Positive Control

2083.1

281.3

616

2872

2372.9

958.4

417

5230

TA 102

Solvent Control

407.1

78.3

129

530

501.2

106.5

192

674

Negative Control

401.5

78.4

197

550

508.0

109.5

160

682

Positive Control

3432.0

1516.2

1183

6415

2278.9

608.0

872

3370

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative