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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EU-Method B.46 in vitro Skin Corrosion: Human Skin Model Test
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name Slags, steelmaking - SMS
Appearance grey granules (soldis)
Molecular formula not applicable (UVCB)
Molecular weight not applicable (UVCB)
Purity 100 w/w % slag
Homogeneity homogenous
Vapour pressure extremely low (melting point >300°C)
Stability solid slag is stable at room temperature
Solubility slightly soluble in water
Production date not stated (2009)
Expiry date 12/2024
Storage Room Temperature: (20 ± 5°C)

Test animals

Species:
other: Homo sapiens (skin)
Strain:
other: not applicable
Details on test animals and environmental conditions:
not applicable since Human Skin Model Test was performed. This test uses commercially available Epi-200-Kit.
The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous, and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.
Epi-200-Kit and MTT-100 assays diluent were obtained from MatTek Corporation in Ashland, USA.

Test system

Type of coverage:
other: in vitro
Preparation of test site:
other: not applicable
Vehicle:
water
Controls:
not required
Amount / concentration applied:
1 mg/ml of the test item/well
Duration of treatment / exposure:
1 h
Observation period:
1 h
Number of animals:
not applicable
Details on study design:
The following media were obtained from MatTek Corporation (description from LAUS GmbH)
MTT Medium
Contains 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, which can be re-duced to a blue formazan
DPBS-Buffer
Solution for the rinsing of the tissues
Assay Medium
Serum-free DMEM medium
Test was performed in several well-plates to account for positive controls (Sodium dodecylsulphate) and negative controls (Dulbecco’s Phosphate Buffered Saline ) (The exact conduct of the test is described in the original study report)
For each experiment, eight 6-well-plate are prepared as holding plate. 3 wells of each plate are filled with 0.9 mL assay medium, the other 3 with 0.9mL MTT medium. The plates are stored in the incubator for 18 hours.
After pre-incubation, the assay medium is replaced by fresh assay medium and the test is started, using one plate (three tissues) as negative control each tissue treated with 30 µL Dulbecco’s Phosphate Buffered Saline, one plate (three tissues) as positive controls each tissue treated with 30 µL SDS-solution and one plate (three tissues) for testing the test item.
After the respective incubation time, the inserts are removed from the plates and are thoroughly rinsed with PBS, blotted and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they are immediately moved to the wells containing MTT solution, blotting the bottom again before setting the insert into the MTT well. The tissues are incubated with MTT medium for three hours and the MTT medium is replaced by PBS buffer. This is then replaced several times. At last, each insert is thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol are pipetted. The plate is then left to stand over night at room temperature. The inserts in which formazan has been produced over night are extracted.
From each well, three replicates with 200 µL solution (each) are pipetted into a 96-well-plate which is read in a plate spectral photometer at 570 nm.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
116
Remarks on result:
other:
Remarks:
Basis: other: Human Skin Model. Time point: 1 h. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:
Comparison of Formazan Production
For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:
% Formazan Production
Designation Slag, Positive Control
steelmaking - SMS,
fine-ground (< 90 µm)
% Formazan production (Tissue 1) 111.7% 9.9%
% Formazan production (Tissue 2) 119.2% 10.7%
% Formazan production (Tissue 3) 117.0% 12.2%
% Formazan production Mean 116.0% 10.9%

Other effects:
no effects caused by slag

Any other information on results incl. tables

 Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Table1 Absorption values blank isopropanol (OD at 570 nm)

Replicate

1

2

3

4

5

6

7

8

Mean

Absorption

0.077

0.097

0.075

0.084

0.088

0.121

0.130

0.119

0.099

 

The absorption values of negative control, test item and positive control are given in the following table:

Table2 Absorption Values negative control, test item and positive control (OD at 570 nm) 

Designation

Measurement

Negative Control

Slag,

steelmaking - SMS,

fine-ground (< 90 µm)

Positive Control

Tissue 1 

1

1.265

1.557

0.229

2

1.343

1.650

0.234

Tissue 2 

1

1.414

1.693

0.238

2

1.410

1.716

0.247

Tissue 3 

1

1.607

1.678

0.267

2

1.634

1.672

0.261

 

From the measured absorptions, the mean of each tissue was calculated, subtracting the mean absorption of isopropanol as given in table 1. Mean and relative standard deviation (comparison of the three tissues) were also calculated.

Table3 Mean Absorption Values

Designation

Negative Control

Slag,

steelmaking - SMS,

fine-ground (< 90 µm)

Positive Control

Mean – blank (Tissue 1)

1.205

1.505

0.133

Mean – blank (Tissue 2)

1.313

1.606

0.144

Mean – blank (Tissue 3) 

1.522

1.576

0.165

Mean of the three Tissues

1.347

1.562

0.147

Relative Standard Deviation
of the three tissues

12.0%

3.3%

11.1%

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
SMS: Human Skin Model Test (in vitro): not irritating
Executive summary:

One valid experiment was performed.

Three tissues of the human skin model EpiDermTMwere treated withSlag, steelmaking - SMS, fine-ground (< 90 µm)for 60 minutes.

30 µL of the eluate of the test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size.

DPBS-buffer was used as negative control, 5% SDS-solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18%).

After the treatment with the test item, the relative absorbance values were increased to 116 %. This value is well above the threshold for irritation potential (50%). Therefore,Slag, steelmaking - SMS, fine-ground (< 90 µm)is considered as

not irritant in the Human Skin Model Test.