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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8.4.2010-9.4.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: MatTek Ocular Irritation Protocol: Neat Method (MTT ET-50), Rev. 1/1/2001
Deviations:
yes
Remarks:
A 1:1 slurry/paste of material and H2O for injection has been prepared as an application form of the test substance. The mixture was very slurry so it could not be applied with the aid of application pin (a deviation from Study Plan and MatTek instruction
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Slimes and Sludges, blast furnace and steelmaking
EC Number:
266-006-2
EC Name:
Slimes and Sludges, blast furnace and steelmaking
Cas Number:
65996-73-8
Molecular formula:
not applicable
IUPAC Name:
Slimes and sludges, blast furnace and steelmaking
Details on test material:
- Physical state: solid
- Composition of test material, percentage of components: Fe (total) 54.29% (mainly as oxides), CaO 10.17%, Zn 5.25%, MgO 4.32%, C 2.06%, SiO2 1.64%, Mn 0.69%, K2O 0.126%, Na2O 0.239%, Al2O3 0.16%
- Lot/batch No.: 21.10.2009
- Expiration date of the lot/batch: unlimited
- Stability under test conditions: unlimited
- Storage condition of test material: stored in PE container at room temperature

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
TISSUES:
The reconstructed human epidermal corneal tissue EpiOcular(OCL-200, MatTek, Ashland, USA); lot No. 13126, kit A.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
3, 30 and 60 minuts
Number of animals or in vitro replicates:
3
Details on study design:
TEST SYSTEM:
MatTek's EpiOcular corneal model (OCL-200, MatTek, Ashland, USA) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. The epidermal cells, which are cultured on specially prepared cell culture inserts using serum free medium, differentiate to form a multi-layered structure which closely parallels the corneal epithelium. The EpiOcular System is manufactured according to defined quality assurance procedures.

TEST METHOD:
The test consists of a topical exposure of the test chemical to a reconstructed human corneal model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with PBS) is used to predict the skin irritation potential.
Evaluation is determined by measuring of optical density (OD) of the formazan extracts using a spectrophotometer at 570 nm. Viability percentage is calculated fort each application time. The % viability (linear y axis) is then plotted versus the dosing time (log x axis). ET-50 value is calculated from slope of the line obtained.

TEST PROCEDURE:
a) Test substance application:
100 mg of the test substance was applied onto previously moistened (100 µL of sterile distilled water) tissue. At application it was assured that the tissues were completely covered by the test substance.
b) Direct MTT reduction:
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional checks for this possibility was performed as follows: 25 mg of the test substance was added to 1 ml MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 60 min. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.
c) Procedure:
On the day of receipt, EpiDerm tissues were conditioned for one hour by incubation to release transport stress related compounds and debris. After pre-incubation, tissues were topically exposed to the test chemical for 3, 30 and 60 minutes. Three tissues were used per each time interval three for the positive control (PC) and three for negative control (NC). Tissues are then thoroughly rinsed, blotted to remove the test substances, and transferred to 5 ml of fresh medium. After 10 minutes of soaking the tissues were transferred to 24-well plates containing MTT medium (1 mg/ml). After 3 hr MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted overnight, without shaking with 2.0 ml/tissue of isopropanol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm.
d) OD570 measuring:
OD570 is measured on a spectrophotometer Libra S22. Isopropanol serves as a blank.

ACCEPTANCE CRITERIA:
a) Negative Control:
The absolute OD of the negative control (NC) tissues (treated with sterile PBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is = 1.0 and = 2.5.
b) Positive Control:
A 0.3% Triton X-100 solution is used as positive control (PC) and tested concurrently with the test chemicals.
The assay meets the acceptance criterion if the ET-50 of PC is between 15 and 45 min.
c) Inter tissue viability difference:
Since in each test eye irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.
The assay meets the acceptance criterion if difference among viabilities of three identically treated tissues <30% (i.e. average ±15%). Single tissue lying out of the interval can be omitted. In the case of two outliers, the substance should be re-tested.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: % tissue viability
Run / experiment:
60 min
Value:
63.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: % tissue viability
Run / experiment:
30 min
Value:
64.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: % tissue viability
Run / experiment:
3 min
Value:
86.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
see table 1

Any other information on results incl. tables

Table 1: Results of MTT-test

time (min) treatment  OD570 values   mean SD % NC
1 2 3
60 negative control  water 1.882 14.717 1.815 1.805 0.068 100.0 
3 C1  106/09 1.541 1.538 1.621 1.567 0.038 86.8
30 C1  106/09 0.946 1.143 1.411 1.167 0.191 64.6
60 C1  106/09 1.041 1.289 1.130 1.153 0.103 63.9
15 positive control  0.3% Triton X-100 1.165 1.661 1.070 1.118 0.259 61.9
45 positive control  0.3% Triton X-100 0.432 0.463 0.487 0.461 0.023 25.5

data in grey-coloured windows were excluded from evaluation

Table 2: Corellation of ET-50 with Draize score and classification

Draize Score   Irritancy Classification   EpiOcular ET-50 (min)
0-15 Non-irritating, Minimal >60
15.1-25 Mild  30-60
25.1-50 Moderate 3-29.99
50.1-110 Severe, Extreme <3

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, the test substance, Slimes and Sludges, blast furnace and steelmaking, was non-irritating for EpiOcular tissues.
Executive summary:

Test substance Slimes and Sludges, blast furnace and steelmaking was assayed for the in vitro eye irritation in human corneal model EpiOcularTM. The test was performed according to MatTek Ocular Irritation Protocol: Neat Method (MTT ET-50), Rev. 1/1/01 (1).

The test substance was applied in delivered form onto a tissue moistened with water. Length of exposition was 3, 30 and 60 minutes. Three tissues were used for each time interval, six for positive control (PC) and three for negative control (NC).

After rinsing and soaking in medium, tissues were incubated with MTT for three hours and then extracted overnight without shaking. OD570 of isopropanol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each three tissues as % of the mean viability of the negative control. A chart of semi-log scale - the % viability (linear y axis) versus the dosing time (log x axis) was constructed and ET-50 value was calculated from slope of the line obtained. The ET-50 calculated was 255.4 min. It responses to the Draize score 0-15 and to the Kay and Calandra classification category non irritant.

In the experiment arrangement given above, the test substance Slimes and Sludges, blast furnace and steelmaking was non irritating in EpiOcular model.