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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:
from 15 August 2002 to 14 October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production bureau, November 24, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): DV6850
- Substance type: powder
- Physical state: solid
- Analytical purity: 83.7%
- Lot/batch No.: R0332-52C
- Expiration date of the lot/batch: 2003/09/30
- Storage condition of test material: ca 4°C in the dark in dry conditions

Method

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 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
supplemented liver homogenate fraction (S9 mix)
Test concentrations with justification for top dose:
test 1 (range-finding): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
test 2: 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solubility of DV6850 was assessed at 50 mg/mL in water, in which it dissolved and formed a clear gel after sonication for 30 minutes. Water (purified in-house by reverse osmosis) was, therefore, used as the solvent for this study.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Used in the absence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Used in the absence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Used in the absence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2)
Remarks:
Used in the absence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Used in the presence of S9 mix
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Used in the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, test 1) and preincubation (30 min pre-incubation, test 2)

DURATION
- Preincubation period: 30 minutes in second test, no preincubation in first test
- Exposure duration: ca. 72 hours

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls with some evidence of a positive dose-relationship (increased revertant colony counts at concentrations below that at which the maximal increase is obtained), it will be considered to show evidence of mutagenic activity in this test system. No statistical analysis was performed.
If exposure to a test substance does not produce an increase in revertant colony numbers in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.

Statistics:
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after the additional testing outlined in the mutation test procedure, the test data may have been subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are ususally analysis of variance followed by Dunnett's test. Biological significance should always be considered along with statistical significance.

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 applicable
Positive controls validity:
valid
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic effects were observed at 5000 µg/mL without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
MAIN ASSAYS:
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to DV6850 at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to DV6850.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 - Results for test 1 (range-finding)

Metabolic activation

Test group

Dose level [µg/plate, for water: mL/plate]

Revertant colony counts (mean±SD)

TA98

TA100

TA1535

TA1537

WP2uvrA

yes

DV6850

5000

57±5

161±13

17±3

26±2

214±54

yes

DV6850

1500

66±5

169±5

18±7

27±5

180±28

yes

DV6850

500

55±15

182±16

14±4

26±5

181±17

yes

DV6850

150

57±2

172±21

14±5

21±4

186±4

yes

DV6850

50

62±13

165±13

16±3

24±6

199±16

yes

DV6850

15

61±16

158±10

17±3

27±9

169±22

yes

DV6850

5

59±8

174±14

20±2

23±7

166±15

yes

water

0.1

60±10

192±27

24±3

19±8

152±8

no

DV6850

5000

38±12

150±11

26±3

13±5

127±43

no

DV6850

1500

41±4

156±14

22±4

18±3

203±14

no

DV6850

500

44±12

149±4

19±2

24±2

184±40

no

DV6850

150

52±4

155±16

10±4

10±3

194±26

no

DV6850

50

56±7

145±6

14±10

10±4

159±11

no

DV6850

15

54±11

148±6

22±9

7±2

169±7

no

DV6850

5

50±14

119±18

22±6

13±5

135±56

no

water

0.1

46±8

157±12

19±4

22±6

199±20

yes

Benzo(a)pyrene

5

666±87

743±8

 

350±8

 

yes

2-Aminoanthracene

2

 

 

65±5

 

 

yes

2-Aminoanthracene

10

 

 

 

 

601±563

no

2- Nitrofluorene

1

208±14

 

 

 

 

no

Sodium azide

0.5

 

588±32

405±15

 

 

no

9-Aminoacridine

30

 

 

 

273±88

 

no

AF-2

0.05

 

 

 

 

841±80

Table 2 - Results for test 2, with pre-incubation

Metabolic activation

Test group

Dose level [µg/plate, for water: mL/plate]

Revertant colony counts (mean±SD)

TA98

TA100

TA1535

TA1537

WP2uvrA

yes

DV6850

5000

42±7

201±18

12±4

42±10

206±14

yes

DV6850

1500

39±11

187±31

23±1

42±12

140±56

yes

DV6850

500

49±13

161±11

19±3

31±3

215±9

yes

DV6850

150

47±5

173±16

21±2

23±12

211±36

yes

DV6850

50

51±12

191±21

17±2

49±10

232±21

yes

water

0.1

49±12

180±2

20±13

30±3

206±11

no

DV6850

5000

38±5

187±14

23±6

30±6

118±10

no

DV6850

1500

36±7

199±15

15±6

31±3

166±16

no

DV6850

500

33±4

182±21

20±5

27±3

136±3

no

DV6850

150

35±5

193±23

14±5

29±6

157±7

no

DV6850

50

39±4

172±12

14±4

24±5

162±27

no

water

0.1

34±10

173±18

12±3

31±7

135±23

yes

Benzo(a)pyrene

5

616±32

583±57

 

136±82

 

yes

2-Aminoanthracene

2

 

 

354±219

 

 

yes

2-Aminoanthracene

10

 

 

 

 

1149±119

no

2- Nitrofluorene

1

204±59

 

 

 

 

no

Sodium azide

0.5

 

369±49

111±79

 

 

no

9-Aminoacridine

30

 

 

 

690±175

 

no

AF-2

0.05

 

 

 

 

1566±121

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that, under the test conditions employed, DV6850 showed no evidence of mutagenic activity in this bacterial system.
Executive summary:

The mutagenic potential of DV6850 in a bacterial system was assessed in a valid GLP study. The study was conducted in compliance with the OECD Guideline 471: Bacterial Reverse Mutation Test.

In this in vitro assessment of the mutagenic potential of DV6850, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA 1535, TA 1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to DV6850 diluted in water. Water was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254—treated rats (S9 mix). The first (range-finding) test was a standard plate incorporation assay, the second involved a pre-incubation stage.

Concentrations of DV6850 up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. Slight cytotoxic effects were observed for E. coli treated at 5000 µg/mL without metabolic activation. No other signs of toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of DV6850 in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, DV6850 showed no evidence of mutagenic activity in this bacterial system.