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Genetic toxicity of ferrous slags was tested in two types of in vitro tests:

Ames-Test

For determination of the mutagenic potential of ferrous slag, the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 was performed with ABS, EAF C and EAF S. As the substance is an inorganic solid, a leachate of the slag was prepared as prescribed by DIN 38414-S (100 g/L).

Two experiments were performed for each slag:

In the first experiment, 5 concentrations of the leachate (ranging from approximately 10000 to 100 µg/plate, nominal concentration) were used. 5 genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the slag both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method.

In the second experiment, 5 concentrations of the leachate (ranging from approaximately 10000 to 600 µg/plate) were tested using the pre-incubation method.

Both experiments yielded the same results: None of the concentrations caused any significant increase in the number of revertant colonies in the tested bacterial strains. The slag had no mutagenic effect towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535. The slag exhibited no sign of toxicity towards the bacteria. No inconsistencies were observed in the sterility control and in the determination of the titre. The numbers of spontaneous revertants of the negative controls were in the normal range, and all positive controls showed mutagenic effects with and without metabolic activation.

ABS, EAF C and EAF S are not mutagenic under the conditions of the test (LAUS 2010b, c and d).

For determination of the mutagenic potential of BOS and SMS, the Bacterial Reverse Mutation Test following OECD 471 was performed. As the slags are inorganic solids, leachates of the slags were prepared with LS (liquid/solid) of 1:4 (250 g/L). Two experiments were performed:

In the first experiment, 5 concentrations of the leachate (8, 40, 200, 1000, 5000 µg/plate, nominal concentrations) were used. 1 genetically manipulated strain of Salmonella typhimurium (TA 100) was exposed to the slag leachates both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method.

In the second experiment, 5 concentrations of the leachate (approximately 640, 1080, 1800, 3000, 5000 µg/plate) were tested using the pre-incubation method with both TA 98 and TA 100.

Both experiments yielded the same results: None of the concentrations caused any significant increase in the number of revertant colonies in the tested bacterial strains. The slags had no mutagenic effect towards Salmonella typhimurium, strains TA 98 and TA 100. The slag exhibited no sign of toxicity towards the bacteria. No inconsistencies were observed in the sterility control and in the determination of the titre. The numbers of spontaneous revertants of the negative controls were in the normal range, and all positive controls showed mutagenic effects with and without metabolic activation.

BOS and SMS are neither mutagenic nor cytotoxic and do not need to be classified as mutagenic. No signal word and no hazard statement is required (Reis da Silva et al. 2006, 2007).

HPRT-Test

To investigate the cytotoxic and mutagenic potential of ferrous slags, a test according to the EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test) was performed with slags, ferrous metal, blast furnace (air cooled - ABS), granular (8-11 mm). The investigation tests the potential of the slag applied as DIN 38414 S4 leachates, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

Two independent experiments were performed, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation, up to 10 % v/v of the undiluted extract being the maximum possible concentration of aqueous extracts in the HPRT test system.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in all experiments. The sensitivity of the test system, and the effectiveness of the metabolic activation system, was confirmed by tests with reference mutagens, used as positive controls.

Slags, ferrous metal, blast furnace (air cooled - ABS), granular (8-11 mm) did not induce gene mutations at the HPRT locus in V79 cells. Slags are neither cytotoxic nor mutagenic under the conditions of the HPRT (Hypoxanthin-Guanin-Phosphoribosyltransferase) gene mutation test according to EU 440/2008 B.17 (Harlan 2009).

 

Mammalian Chromosome Aberration Test

For determination of the mutagenic potential of ferrous slag, the in-vitro Test “Chromosome Aberration in Human Lymphocytes” following OECD 473 and EU B.10 was performed with ABS, granular (8-11 mm) eluate dissolved in water in absence and presence of an exogenous metabolic activation system.Two independent experiments were performed. In each experimental group, all cell cultures were set up in duplicates. In both independent experiments, no toxicity was detected and neither a statistically sig-nificant nor a biologically relevant increase of structural chromosomal aberrations was ob-served at the evaluated concentration. Furthermore, no increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the fre-quencies of the controls.

All positive control compounds caused large, statistically significant increases in the pro-portion of aberrant cells, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported, Slag, ferrous metal, blast furnace did not in-duce structural chromosomal aberrations in human lymphocytes in vitro.


Short description of key information:
Ferrous slags do not have any mutagenic potential in the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14.
Slag, ferrous metal, blast furnace (air cooled - ABS), granular (8-11 mm) is neither cytotoxic nor mutagenic under the conditions of the HPRT (Hypoxanthin-Guanin-Phosphoribosyltransferase) gene mutation test according to EU 440/2008 B.17.
Slag, ferrous metal, blast furnace (air cooled - ABS), granular (8-11 mm) is not mutagenic under the conditions of the “Chromosome Aberration in Human Lymphocytes”-Test following OECD 473 and EU B.10

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All tests performed with ferrous slags were negative, and there is no indication, that any slag has any mutagenic or any relevant cytotoxic potential.