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Toxicological information

Genetic toxicity: in vitro

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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:
2005-08-22 to 2005-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005

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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
see Table 1
Vehicle / solvent:
- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: DMSO is a common vehicle for the Ames test.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylene-diamine
Remarks:
TA97a without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
TA97a with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 and TA1535 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
TA98, TA100 and TA1535 with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: t-Butyl-hydroperoxide
Remarks:
TA102 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxy-anthraquinone
Remarks:
TA102 with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 2 days


NUMBER OF REPLICATIONS: triplicate for each dose group, six replicates for the solvent controls and three replicates for positive control.



DETERMINATION OF CYTOTOXICITY

- Method: other: reduced or no bacterial background lawn, microcolonies of bacteria instead of a homogenous background lawn, clearly reduced number of revertant colonies.

Evaluation criteria:
A result would be considered positive if there was a reproducible increase in the number of revertants to more than 2.5 fold for strains TA98 and TA1538 and more than 1.33 fold for TA97a, TA100 and TA102.
Statistics:
The number of revertant colonies was counted by hand in strains TA98 and TA1535 and by a computer program in strains TA97a, TA100 and TA102. The mean and standard deviation of replicate results were calculated.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
185 μg/plate to strain TA97a
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: A precipitate was visible at 556 μg/plate samples and above. The precipitate was still visible at 5000 μg/plate when the colonies were counted but did not impede the counting.

- Other confounding effects: the substance is known to be ready biodegradable.

RANGE-FINDING/SCREENING STUDIES: see Table 2


COMPARISON WITH HISTORICAL CONTROL DATA: numbers of spontaneous revertants are comparable with the historic control data for the negative controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Dose range-finding study. Number of revertants per plate (average of 2 plates).

 

TA 100

Concentration (μg/Plate)

- MA

Cytotoxic (Yes/No)

21

93

No

62

93

No

185

81

No

556

58

No

1667

59

No

5000

75

No

 

 

Table 3: Experiment 1 Mutagenicity Assay. Number of revertants per plate (mean of 6 plates in solvent control, mean of 3 plates in the other exposure).

 

TA97a

TA98

TA100

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

5000

-

-

Yes

6.0

9.0

No

42.7

46.3

No

1667

0.0

9.7

Yes

8.7

9.3

No

44.3

48.0

No

556

0.0

54.7

Yes

6.3

8.0

No

52.0

56.3

No

185

67.7

110.3

No

11.0

11.0

No

66.3

69.7

No

62

119.3

120.3

No

9.3

9.3

No

66.0

79.0

No

0*

607.0

140.5

No

8.0

12.8

No

73.5

77.0

No

Positive control

374.0

607.0

No

109.7

415.7

No

422.3

1955.0

No

*solvent control with DMSO

 

Table 3 (continued): Experiment 1 Mutagenicity Assay. Number of revertants per plate (mean of 6 plates in the solvent control, mean of 3 plates in the other exposures).

 

TA102

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

5000

66.3

127.0

Yes

7.0

4.3

Yes

1667

85.0

152.7

Yes

7.3

7.7

Yes

556

102.7

198.0

No

11.0

10.7

No

185

117.0

244.3

No

22.0

16.3

No

62

170.3

260.7

No

17.7

18.7

No

0*

181.7

247.8

No

20.0

18.2

No

Positive control

487.7

595.0

No

207.0

196.7

No

*solvent control with DMSO

Table 4: Experiment 2 Mutagenicity Assay. Number of revertants per plate )mean of 6 plates per solvent control, mean of 3 plates in other exposures).

 

TA97a

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(
yes/no)

556

4.3

8.3

Yes

185

32.7

102.7

No

62

102.3

108.3

No

21

110.0

103.7

No

7

109.3

135.0

No

2.3

118.0

122.7

No

0*

85.8

110.7

No

Positive control

229.7

351.7

No

*solvent control withDMSO

Table 4 (continued): Experiment 2 Mutagenicity Assay. Number of revertants per plate (mean of 6 plates per solvent control, mean of 3 plates in other exposures).

 

TA98

TA100

TA100

Conc.
(
µg/plate)

— MA

+MA

Cytotoxic
(
yes/no)

MA

+MA

Cytotoxic
(
yes/no)

MA

+MA

Cytotoxic
(
yes/no)

5000

6.3

7.3

No

54.7

56.0

No

54.7

56.0

No

1667

5.7

6.7

No

57.3

61.3

No

57.3

61.3

No

556

5.3

8.0

No

59.0

68.0

No

59.0

68.0

No

185

5.7

10.7

No

69.0

79.0

No

69.0

79.0

No

62

5.3

11.3

No

69.0

77.0

No

68.0

77.0

No

0*

7.7

11.8

No

86.5

69.5

No

86.5

69.5

No

Positive control

207.3

359.3

No

253.0

1372.0

No

253.0

1372.0

No

*solvent control withDMSO

Table 4 (continued): Experiment 2 Mutagenicity Assay. Number of revertants per plate (mean of 6 plates per solvent control, mean of 3 plates in other exposures).

 

TA102

TA1535

Conc.
(
µg/plate)

MA

+MA

Cytotoxic
(
yes/no)

MA

+MA

Cytotoxic
(
yes/no)

5000

34.7

85.3

Yes

5.0

4.7

Yes

1667

45.3

100.3

Yes

6.3

7.3

Yes

556

51.3

125.7

Yes

10.7

12.0

No

185

80.0

149.7

No

15.0

17.0

No

62

86.0

166.3

No

183

19.7

No

0*

112.5

147.8

No

15.7

17.7

No

Positive control

344.0

615.0

No

158.0

163.0

No

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance did not produce an increase in the number of revertants in S. typhimurium (strains TA97a, TA98, TA100, TA102 and TA1535) when tested under GLP to OECD 471 (2000). The test material was therefore considered to be non-mutagenic under the conditions of this test.