Slimes and Sludges, blast furnace and steelmaking

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Test substance Slimes and Sludges, blast furnace and steelmaking was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity - Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471. To investigate the potential of Slimes and Sludges, blast burnace and steelmaking to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out. The test was performed according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test). The test item Slimes and Sludges, blast furnace and steelmaking was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The test was performed according to OECD Guideline for Testing of Chemicals, No. 476, In vitro Mammalian Cell Gene Mutation Tests.

In vivo Mammalian Erythrocytes Micronucleus Test was performed in accordance with OECD TG 474. All the above mentioned in vitro tests were practised in accordance with GLP.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Velaz, s.r.o., Kolec u Kladna, Czech Republic (SPF breeding)
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: Males (M) 210.5 - 315.3 g; Females (F) 160.4 - 223.6 g
- Assigned to test groups randomly: yes
- Housing: cages with sterilized shavings of soft wood, monitored conditions, microbiologically defined background
- Diet (e.g. ad libitum): pelleted standard diet for experimental animals ST 1 BERGMAN ad libitum, manufacturer: Ing. Miroslav Mrkvicka – Výroba krmných smesí, Mlýn Kocanda 19, Jesenice u Prahy, Czech Republic
- Water (e.g. ad libitum): drinking tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C, monitored permanently
- Humidity (%): 30 – 70 %, monitored permanently
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: chosen with respect to previous experience with testing of test item testing facility
- Concentration of test material in vehicle: adjusted at all dose levels, in order constant volume of the application form - 2 ml/100 g of body weight
- Amount of vehicle (if gavage or dermal): adjusted at all dose levels and control, in order constant volume of the application form - 2 ml/100 g of body weight
- Lot/batch no. (if required): 5211201 (supplier: Dr. Kulich, Pharma, s.r.o., Czech Republic)

Details on exposure:

Test item was administered in olive oil by gavage using a stomach tube. Negative control (olive oil only) was administered in the same way. Olive oil as vehicle was chosen with respect to previous experience with testing of test item in our facility. Positive control was administered by intraperitoneal injection (solution in water for injection). Both routes of administration are listed in guideline.
The volume of the application form at all dose levels and negative control was constant - 2 ml/100 g of body weight by adequate adjusting the concentration. The animals were weighed before application. The application form of the test substance was prepared immediately before administration.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Remarks:

Doses / Concentrations:
2000 mg/kg body weight
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg body weight
Basis:
actual ingested

Remarks:

Doses / Concentrations:
500 mg/kg body weight
Basis:
actual ingested

No. of animals per sex per dose:

2000 mg/kg, exposition – 24 h: 5 males + 5 females
2000 mg/kg, exposition – 48 h: 5 males + 5 females
1000 mg/kg, exposition – 24 h: 5 males + 5 females
1000 mg/kg, exposition – 48 h: 5 males + 5 females
500 mg/kg, exposition – 24 h: 5 males + 5 females
500 mg/kg, exposition – 48 h: 5 males + 5 females
negative control – olive oil, time interval – 24 h: 5 males + 5 females
negative control – olive oil, time interval – 48 h: 5 males + 5 females
positive control – cyclophosphamide 20 mg/kg, time interval – 24 h: 5 males + 5 females
control without administration: 5 males + 5 females

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

Positive control was administered Cyclophosphamide monohydrate (CAS No.: 6055-19-2) by intraperitoneal injection (solution in water for injection). A dose level for cyclophosphamide - 20 mg/kg of body weight - was chosen on the base of literature data.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Evaluation of cytotoxicity of the test item was performed according to comparison of cytotoxicity indexes. Index of the dose level of 2000 mg/kg was not statistically significantly changed compared to control animals and was comparable to control group. On the basis of the results from the pilot experiment the concrete dose levels for the main study were determined: 2000 mg/kg, 1000 mg/kg, 500 mg/kg.

DETAILS OF SLIDE PREPARATION:
Bone marrow cells were obtained from the femora immediately after euthanasia of animals.
After excising and careful cleaning of the bone both femur ends were clipped off. Marrow was gently flushed from the bone by 1 ml of bovine serum into the tube. Acquired bone marrow was mixed several times by syringe with thin needle.
The bone marrow with serum in tubes was centrifuged (5 min – 1000 rpm). The supernatant was gently removed, one drop of bovine serum was added to the sediment and this cell suspension was mixed on mixer. Clean and degreased slides were marked by the number of study, number of animal, sex and dose level. One drop of cell suspension was placed onto the slide and a smear was prepared using a pusher slide. Two bone marrow smears were prepared per animal.
After drying (20 minutes, 60°C) the smears were fixed by ethanol – 5 minutes. Then they were twice rinsed by distilled water and stained by 5% solution of Giemsa – 15 minutes.

METHOD OF ANALYSIS:
Determined parameters:
1) the number of immature erythrocytes among 200 total RBC per animal, which provides data for calculation of an index of bone marrow cytotoxicity
2) the number of micronucleated positive polychromatic erythrocytes among total 2000 polychromatic erythrocytes per animal (which provides information about chromosomal damage).
Slides of bone marrow cells were coded before scoring. After the analysis was completed, the slides were decoded and the data summarized.

Criteria for distinguishing the micronuclei:
- colour (purple)
- form (generally round or almond shaped, occasional lightly stained and ring shaped micronuclei may occur)
- borders (sharp)
- size (5-20% of polychromatic erythrocyte size)

Scoring of micronucleated immature erythrocytes:
- number of immature erythrocytes with micronuclei (number of micronuclei per PE is not a result, occasionally more than one micronucleus may appear per PE)

Evaluation criteria:

- Positive result: there is a statistically significant increase in micronucleated polychromatic erythrocytes at one or more dose levels, and this increase is dose-related
- Negative result: none of the criteria for positive response are met
- Equivocal result: when the test results cannot be identified clearly as positive or negative

Statistics:

The statistical analysis was performed by the software ANOVA - Analysis of Variance (software QC.Expert 2.5, Trilobyte, Statistical Software, Czech Republic) for identification of factor influence. Subsequently the Scheffé´s method was used for pair comparison of means. Each of treatment groups was confronted with negative control group.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY
The experiment was performed because there were no suitable data, which could be used for the dose selection. Pilot test was performed in the same laboratory, using the same strain of animals and the same treatment regimen that was used in the main study. The test item was administered by gavage using a stomach tube.

Dose levels in pilot experiment and duration of exposition:
2000 mg/kg of body weight – exposition – 24 h: 3 animals
2000 mg/kg of body weight – exposition – 48 h: 3 animals
negative control – olive oil - exposition – 24 h: 3 animals

Clinical observation:
No clinical symptoms of toxicity were observed in animals of all groups.

Dose level determination:
Evaluation of cytotoxicity of the test item was performed according to comparison of cytotoxicity indexes. Index of the dose level of 2000 mg/kg was not statistically significantly changed compared to control animals and was comparable to control group. On the basis of the results from the pilot experiment the concrete dose levels for the main study were determined: 2000 mg/kg, 1000 mg/kg, 500 mg/kg.

Table 1: Micronucleated immature erythrocytes (IME)

Group	Number of micronucleated IME per animal (per 2000 IME)		Percentage expression
	Males	Females	Males	Females
2000 mg/kg – 24 h	2.18 ± 0.83	1.77 ± 0.84	0.109 ± 0.04	0.089 ± 0.04
1000 mg/kg – 24 h	1.58 ± 1.12	2.17 ± 0.83	0.079 ± 0.06	0.109 ± 0.04
500 mg/kg – 24h	1.78 ± 0.82	2.17 ± 0.82	0.089 ± 0.04	0.109± 0.04
Negative control – 24 h	2.18 ± 0.83	1.98 ± 0.99	0.109 ± 0.04	0.099 ± 0.05
2000 mg/kg – 48 h	1.78 ± 0.82	1.98 ± 0.70	0.089 ± 0.04	0.099 ± 0.03
1000 mg/kg – 48 h	2.18 ± 0.83	2.18 ± 0.82	0.109 ± 0.04	0.109 ± 0.04
500 mg/kg – 48 h	2.18 ± 0.83	1.98 ± 0.70	0.109 ± 0.04	0.099± 0.03
Negative control – 48 h	2.18 ± 0.83	1.98 ± 0.69	0.109 ± 0.04	0.099± 0.03
Positive control – 24 h	12.87 ± 1.91	15.66 ± 2.11	0.643 ± 0.10	0.783± 0.11
Control without treatment	2.18 ± 0.83	1.59 ± 1.13	0.109 ± 0.04	0.079± 0.06

Table 2: Cytotoxicity index (Proportion of immature erythrocytes among total erythrocytes)

Group	Males	Females
2000 mg/kg – 24 h	0.402 ± 0.05	0.388 ± 0.05
1000 mg/kg – 24 h	0.391 ± 0.04	0.398 ± 0.05
500 mg/kg – 24h	0.392 ± 0.05	0.395 ± 0.05
Negative control – 24 h	0.398 ± 0.05	0.396 ± 0.05
2000 mg/kg – 48 h	0.398 ± 0.04	0.402 ± 0.05
1000 mg/kg – 48 h	0.387 ± 0.05	0.417 ± 0.05
500 mg/kg – 48 h	0.401 ± 0.04	0.406 ± 0.05
Negative control – 48 h	0.380 ± 0.05	0.398 ± 0.04
Positive control	0.306 ± 0.02	0.305 ± 0.03
Control without treatment	0.406 ± 0.02	0.404 ± 0.05

Conclusions:

Interpretation of results (migrated information): negative
The test item Slimes and Sludges, blast furnace and steelmaking, was tested for the assessment of cytogenetic damage using micronucleus test. In this experimental arrangement, it is not possible to estimate if the test item (or its metabolites) reaches general circulation or specified the target organ. Even the comparison of cytotoxicity indexes between control and treated groups did not provide any information about this event. Statistically significant increase in the number of micronucleated immature erythrocytes compared to the control was not recorded at any dose level.
Under the given test conditions, the test item, Slimes and Sludges, blast furnace and steelmaking, has negative result in micronucleus test.
Negative results in the micronucleus test indicate that under the test conditions, the test item Slimes and Sludges, blast furnace and steelmaking, does not produce micronuclei in immature erythrocytes in bone marrow of rat.

Executive summary:

The test item, Slimes and Sludges, blast furnace and steelmaking, was tested for the assessment of cytogenetic damage in vivo, using laboratory rat (Wistar). The study is a part of the test substance health hazard evaluation. The test was performed according to the EU Method B.12, Mutagenicity – In vivo Mammalian Erythrocyte Micronucleus Test. The method is analogous to the OECD Test Guideline No. 474, Mammalian Erythrocyte Micronucleus Test.

The test item was administered to animals by stomach tube in single dose. Three dose levels were chosen according to the results of pilot experiment - 500, 1000 and 2000 mg/kg of body weight. Two bone marrow sampling intervals were used - 24 and 48 hours after administration. The group of animals without administration and concurrent negative and positive controls were included. Each experimental group consisted of five males and five females. The smears obtained from bone marrow were examined by light microscope. In any of mentioned dose levels the test substance did not cause a significant increase of number of immature erythrocytes with micronuclei in comparison with negative control group.

At given experimental conditions the test item, Slimes and Sludges, blast furnace and steelmaking, did not give rise to formation of micronuclei in immature erythrocytes in bone marrow of rat.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Test substance Slimes and Sludges, blast furnace and steelmaking was assayed for the mutagenicity by the Bacterial Reverse Mutation Test (EU method B.13/14). The test substance Slimes and Sludges, blast furnace and steelmaking was non mutagenic for all the used bacterial strains with as well as without metabolic activation.

During In vitro Mammalian Chromosome Aberration Test (OECD Guideline 473), the test item Slimes and Sludges, blast furnace and steelmaking, increased the number of structural chromosomal aberrations above the historical control data range in presence of the metabolic activation system in the V79 Chinese hamster cell line. Due to the relative moderate effects observed in V79 cells after treatment with the test item, Slimes and Sludges, blast furnace and steelmaking, is considered as equivocal in this chromosome aberration test.

According to results In vitro Mammalian Cell Gene Mutation Tests (OECD guideline 476), the test item Slimes and Sludges, blast furnace and steelmaking is considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

In vivo Mammalian Erythrocytes Micronucleus Test (OECD TG 474) was performed to decide whether tested substance is mutagenic. At experimental conditions of this study the test item, Slimes and Sludges, blast furnace and steelmaking, did not give rise to formation of micronuclei in immature erythrocytes in bone marrow of rat, therefore the test item gave a negative result in micronucleus test.

Based on this results Slimes and Sludges, blast furnace and steelmaking can be considered as non-mutagenic substance.

Justification for classification or non-classification

The three In vitro studies provided ambivalent results. Due to the fact there was decided on the basis of In vivo Mammalian Erythrocytes Micronucleus Test; in accordance with this study can be the Slimes and Sludges, blast furnace and steelmaking considered as non-mutagenic substance.