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Toxicological information

Genetic toxicity: in vitro

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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:
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. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 106cells factor efficiency I efficiency II 106cells 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.