slag, copper smelting

Genetic toxicity: in vitro

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

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

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

Test material information

Constituent 1

Reference substance name:

Iron silicate, copper smelting and refining

EC Number:

701-480-0

Cas Number:

67711-92-6

IUPAC Name:

Iron silicate, copper smelting and refining

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

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

dimethylsulfoxide (DMSO)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

for plates without activation

Controls 2

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.