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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented study performed according to internationally accepted method

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
A bacterial reverse mutation test was performed that used amino-acid requiring strains of Escherichia coli to detect point mutations, which involve substitutions of the genome of the organism. Bacteria was exposed to the test substance with and without metabolic activation.
In addition bacterial survival test was performed that measured the influence of the test substance on the growth of the strain that is used to evaluate the cytotoxicity of the sample
GLP compliance:
no
Remarks:
other quality assurance
Type of assay:
bacterial gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Granulated copper slag with the following chemical composition.
Cu 0.78%
Pb 0.48%
As <0.005%
Ni 0,025 %
Cd 0.006%
Cr 0.25%
Cr VI <0.005%

Method

Species / strain
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Five different conentrations were tested: 312, 625, 1250 , 2500 and 5000 mcg/plate
Vehicle / solvent:
The sample was subjected to an extraction in dimethylsulfoxide (DMSO) Then resulting liquid after filtering is being used to carry out the test
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
dimethylsulfoxide (DMSO)
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
for plates without activation
Untreated negative controls:
yes
Remarks:
dimethylsulfoxide(DMSO)
Positive controls:
no
Positive control substance:
other: 2 Aa- Aminoantraceno
Remarks:
for plates with activation
Details on test system and experimental conditions:
0.1 ml of fresh bacterial culture and and 0.1 ml of DMSO (in the control plates) were added to the different concentrations of the test solution .Approximate cell density of bacterial culture, as measured by colorimetry based on a growth curve was 10~8 cells / mm. To test the effect of enzyme activation the same number of plates were prepared by adding 0.5 ml of enzyme activity in each.
The contents of each tube are mixed and poured over a surface plate contining ET5 media ( E5 media supplemented with tryptophan)
All plates were incubated at 37°+/- 2C for 48 hours. After the incubation period, the number of revertant colonies per plate was counted.
Triplicate plating was used at each dose level.


The survival test was performed by adding 0.1 ml of DMSO or the solution of the sample (at the same concentrations as the test for mutagenicity) and 0.1 ml of bacterial culture (diluted to a density of bacterial population as appropriate) to tubes with 2 agir ml surface.
The plates were incubated 48 h at 37 / - 2C and then number of colonies was counted. All plates were prepared in triplicate.

In parallel tests were performed with MMS( metil metano sulfonad) and 2 Aa-Aminoantraceno) to verify the sensitivity of the strain, following the same methodology.
Evaluation criteria:
Substance is considered as mutagenic if the number of revertant colonies appearing on the treated plates is significantly greater than the number that appeared in the control culture plates.

Substance is considered as cytotoxic if the number of colonies appearing on the treated plates is significantly lower than the number that appeared in the control culture plates.

Results and discussion

Test results
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The results are presented in the tables below. Each column represents the average of the microorganism grown at this concentration .

Statistical comparison of the data in each column was made relative to the control. Muragenicity

 

Without metabolic activation

Plate1

Plate2

Plate3

Media

SD

Control

110

91

84

95

13

312

74

80

85

80

6

625

88

82

75

82

7

1250

122

98

76

99

23

2500

65

82

108

85

22

5000

76

85

72

78

7

 

 

With metabolic activation

Plate1

Plate2

Plate3

Media

SD

Control

138

126

109

124

15

312

135

120

130

128

8

625

130

123

121

125

5

1250

139

117

128

128

11

2500

131

123

113

122

9

5000

112

119

114

115

4

 

Bacteria survival

 

 

Without metabolic activation

Plate1

Plate2

Plate3

Media

SD

Control

66

73

58

66

8

312

73

78

89

80

8

625

73

83

84

80

6

1250

73

99

78

83

14

2500

62

115

75

84

28

5000

78

55

73

69

12

 

 

With metabolic activation

Plate1

Plate2

Plate3

Media

SD

Control

84

76

87

82

6

312

88

76

76

80

7

625

78

75

67

73

6

1250

81

86

81

83

3

2500

77

87

83

82

5

5000

87

63

78

76

12


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

This is a good quality study. Under the conditions of the study, the sample is not mutagenic and does not affect bacterial survival.






Executive summary:

To evaluate the mutagenicity potential of the copper slag a bacterial reverse mutation test was performed with Escherichia coli WP2 uvr A pKM 101 according to EU method B13 "Mutagenicity – reverse mutation test using bacteria"

In addition bacterial survival test was performed that measured the influence of the test substance on the growth of the strain that is used to evaluate the cytotoxicity of the sample

Mutagenicity

Results indicate that at tested concentrations, the increase in the number of revertants compared to the control is not significant. Therefore at these concentrciones the copper slag is considered as non-mutatagenic.

Bacteria survival

Results indicate that at tested concentrations the decrease in the number of colonies comparing to the control is not significant. Therefore at these concentrations the copper slag does not affect bacterial survival.