Ladle skimming slag, Ladle furnace refining, slag, Ferrous slag

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

Study period:

2006-2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2006

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Introduction of reverse mutation in Salmonella typhimurium lineages both in the absence and presence of metabolic activation.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Slag, pig iron ladle refining, non-granulated, water-cooled

Molecular formula:

not applicable because slags are UVCB

IUPAC Name:

Slag, pig iron ladle refining, non-granulated, water-cooled

Details on test material:

In order to substantiate this evaluation, the Tecarn-Tecnologia Ambiental Ltda accredited before the Ministry of Agriculture and is holder of bio-safety certificates.
SMS slag samples were collected in CST´s processing plants. The crushed stone is from Brasitalia quarry, located in the municipality of Serra, Brasilia. Crushed stone samples were collected and prepared by the company Kaerne Engenharia Ltda. In compliance with the method defined in NBR 10.007.2007.

Method

Target gene:

The Ames test employs strains of Salmonella typhimurium derived from the LT2 parental lineage particularly built to detect mutations of reading–table displacement or DNA base pairs replacement types. These strains are unable to grow in minimum culture medium devoid of histidine, unless there are mutations that restore the histidine synthesis capacity.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: mutations hisD3052, uvrB, pKM 101, rfa

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: mutations hisG46, uvrB, pKM 101, rfa

Metabolic activation:

with and without

Metabolic activation system:

S9 liver enzyme mixture used for the test with metabolic activation

Test concentrations with justification for top dose:

A glucosidal minimum medium and surface agar were supplied ready for use. To each test tube containing 3 ml of agar, previously conditioned in a dry bath at 45°C, was added a volume of 0.1 ml of overnight grown bacteria and 0.1 ml of the test substance. For tests with metabolic activation, 0.5 mL/plaque of the S9 fraction was added. This fraction has a protein concentration of 44.4 mg/mL

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Nitroquinoline strain TA98 without S9. Sodium azide, trains TA100 without S9. 2-Amino-anthracene both strains TA98, TA100 with S9.

Details on test system and experimental conditions:

Verification of genetic characteristics:
By growth in a selected medium, the dependence of the bacterial strains on histidine and biotin was checked. The presence of the uvrB deletion was detected by sensitivity to ultraviolet light irradiation. The rfa mutation, which increases permeability of the bacterial wall to facilitate the penetration of test substances, was detected through sensitivity to crystal violet. By testing resistance to ampicillin, the presence of plasmid pKM101 was observed. This plasmid acts at the level of DNA repair, increasing chemical and spontaneous mutagenicity. The number of spontaneously reverting colonies in each strain was determined for comparison with historic controls.
Sample preparation
Since slags are not soluble in water, they were leached. The product was weighted individually in the preparation of each test concentration. The product and water mixtures were maintained under magnetic shaking for 20h and then in rest for 1h of decantation.
Test procedure
A preliminary test was performed with strain TA100 for determination of the appropriate interval or using slag concentrations in the final test and concentrations (8, 40, 200, 1000, 5000 µg/plaque) were used. The concentrations employed in the test were not found to be poisonous to the growth of Salmonella typhimurium. In the final test with strains TA98 and TA100 the following concentrations were used (648, 1080, 1800, 3000, 5000 µg/plaque). All the concentrations were tested in triplicate in both the absence and presence of metabolic activation. The negative controls were performed in triplicate and the positive ones in duplicate in the final test.

Evaluation criteria:

Positive and negative controls were included in all the tests. The negative control employed was deionised water 100 µL/plaque to obtain the number of spontaneously reversing colonies by plaque for subsequent comparison with the number of reverting colonies observed in the presence of the test substance. The positive controls insured the response capacity of each strain to slag and the effectiveness of the metabolic activation.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A result is considered positive when the average number of reversing colonies in the test plaques is equal to or higher than double that observed in the negative control plaque for strains TA98 and TA100. For confirmation of the positive result, the variance analysis must present pANOVA >5% values and the tested concentrations must present a clear concentration-response relationship. Variance analysis indicates the probability that the number of reverting colonies in different conc. is increasing (mutagenity) or decreasing (toxicity).
The test is considered valid when a fine layer of bacterial growth is found in the plaques, the frequency of spontaneous reversion of negative control plaques remains within the range, positive control show mutagenic activity in the tested strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Slags, steelmaking (SMS) and natural stone form a quarry in Serra, Brasilia, are not mutagenetic or cytotoxic under the conditions of the test according to OECD Guideline 471.

Executive summary:

For determination of the mutagenic potential of slags, steelmaking (SMS) and natural stone from a quarry in Serra, Brasilia, the Bacterial Reverse Mutation Test following OECD 471 was performed. As the substance is an inorganic solid, leachates of the slags were prepared with a 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 (648, 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 slag had no mutagenic effect towards Salmonella typhimurium, strains TA 98 and TA 100. The slag exhibited no 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.

Slags, steelmaking (SMS) and natural stone from a quarry in Serra, Brasilia, are not mutagenic or cytotoxic and do not need to be classified as mutagenic. No signal word and no hazard statement is required.