Slags, ferrous metal, blast furnace

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6TG. These cells are able to proliferate in the presence of 6TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The pheno-typic expression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expression period is terminated by adding 6TG to the culture medium

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Slags, ferrous metal, blast furnace

EC Number:

266-002-0

EC Name:

Slags, ferrous metal, blast furnace

Cas Number:

65996-69-2

Molecular formula:

~ Al(n)Ca(m)Mg(o)Si(p)O(3n/2+m+o+2p)

IUPAC Name:

Aluminium-Calcium-Magnesium-Silicium oxide equivalent

Method

Target gene:

HPRT (Hypoxanthin-Guanin-Phosphoribosyltransferase) locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has high proliferation rate (doubling time 12 - 16 h in stock cultures), good cloning efficiency of untreated cells (as a rule more than 50 %), and a stable karyotype with a modal chromosome number of 22

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Vehicle / solvent:

water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

Positive Control Substances
Without metabolic activation
Name: EMS; ethylmethane sulfonate
EINECS: 200-536-7
Supplier: ACROS ORGANICS, 2440 Geel, Belgium
Purity: ≥ 98 %
Dissolved in: Nutrient medium
Final concent.: 0.075 – 0.3 mg/mL = 0.6 – 2.4 mM

With metabolic activation
Name: DMBA; 7,12-dimethylbenz(a)anthracene
EINECS: 200-359-5
Supplier: SIGMA CHEMIE GMBH, 82041 Deisenhofen, Germany
Dissolved in: DMSO, Dimethylsulfoxide;
(E. MERCK, 64293 Darmstadt; Germany
final concentration in nutrient medium 0.5 %)
Final concent.: 1.1 - 2.5 µg/mL = 4.3 – 9.8 µM
The dilutions of the stock solutions will be prepared on the day of the experiment and used immediately.
The stability of both positive control substances in solution is proven by the mutagenic response in the expected range.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results of main experiment (cultures without likelyhood of effect were not continued)

			relative	relative	mutant		relative	relative	mutant
	conc.	S9	cloning	cloning	colonies/	induction	cloning	cloning	colonies/	induction
	%	mix	efficiency I	efficiency II	10^6cells	factor	efficiency I	efficiency II	10^6cells	factor
			%	%			%	%
Column	1	2	3	4	5	6	7	8	9	10
Experiment I / 4 h treatment			culture I				culture II
Solvent control with water		-	100,0	100,0	7,6	1,0	100,0	100,0	17,0	1,0
Positive control with EMS	150 µg/mL	-	112,1	97,0	132,0	17,5	48,5	72,7	291,1	17,1
Test item	1,0	-	111,5	culture was not continued^#			89,1	culture was not continued^#
Test item	2,0	-	119,2	78,8	8,3	1,1	92,2	110,2	13,0	0,8
Test item	4,0	-	106,2	119,1	12,8	1,7	92,5	106,7	12,2	0,7
Test item	6,0	-	116,1	97,3	13,1	1,7	97,9	107,8	14,8	0,9
Test item	8,0	-	110,4	97,8	16,7	2,2	89,3	120,8	15,9	0,9
Test item	10,0	-	126,3	99,5	15,9	2,1	91,9	123,7	18,8	1,1
Solvent control with water			100,0	100,0	14,0	1,0	100,0	100,0	13,9	1,0
Positive control with DMBA	1.1 µg/mL	+	40,6	44,0	1348,1	96,5	48,8	48,1	1031,0	74,3
Test item	1,0	+	98,9	culture was not continued^#			117,2	culture was not continued^#
Test item	2,0	+	100,4	88,2	7,3	0,5	126,4	98,2	15,1	1,1
Test item	4,0	+	106,6	97,7	19,9	1,4	123,1	95,6	15,5	1,1
Test item	6,0	+	99,1	90,0	15,2	1,1	123,8	94,1	6,0	0,4
Test item	8,0	+	105,2	95,0	13,6	1,0	120,1	92,9	10,1	0,7
Test item	10,0	+	96,9	93,1	10,7	0,8	120,0	96,7	12,9	0,9
Experiment II / 24 h treatment			culture I				culture II
Solvent control with water		-	100,0	100,0	7,9	1,0	100,0	100,0	12,7	1,0
Positive control with EMS	150 µg/mL	-	91,4	94,1	444,0	56,2	81,6	94,5	593,7	46,9
Test item	1,0	-	77,8	culture was not continued^#			98,4	culture was not continued^#
Test item	2,0	-	95,7	117,3	11,4	1,4	98,5	94,9	23,0	1,8
Test item	4,0	-	95,8	101,8	8,4	1,1	98,3	98,1	22,7	1,8
Test item	6,0	-	92,9	128,1	12,0	1,5	100,8	107,3	25,7	2,0
Test item	8,0	-	95,9	112,8	10,6	1,3	98,8	88,9	23,3	1,8
Test item	10,0	-	95,5	114,2	8,5	1,1	97,2	108,8	14,5	1,1
Experiment II / 4 h treatment
Solvent control with water		+	100,0	100,0	10,5	1,0	100,0	100,0	13,5	1,0
Positive control with DMBA	1.1 µg/mL	+	35,0	72,9	786,1	74,7	45,1	93,7	1596,7	118,4
Test item	1,0	+	91,1	culture was not continued^#			97,8	culture was not continued^#
Test item	2,0	+	74,2	98,5	12,7	1,2	100,7	103,2	19,3	1,4
Test item	4,0	+	82,3	106,7	12,7	1,2	98,6	144,5	17,5	1,3
Test item	6,0	+	66,3	95,6	7,8	0,7	100,1	172,5	11,0	0,8
Test item	8,0	+	79,3	75,8	26,8	2,5	96,7	138,5	17,0	1,3
Test item	10,0	+	67,5	74,7	15,0	1,4	100,1	159,1	22,2	1,6

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Slags, ferrous metal, blast furnace (air cooled - ABS), granular (8-11 mm) are neither cytotoxic nor mutagenic under the conditions of the HPRT (Hypoxanthin-Guanin-Phosphoribosyltransferase) gene mutation test according to EU 440/2008 B.17.

Executive summary:

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.