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Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2015 - 05 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
yes
Remarks:
The deivation was considered not to affect the purpose or integrity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
yes
Remarks:
The deivation was considered not to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Slags, sponge iron production by coal reduction in retort
EC Number:
940-884-3
Molecular formula:
Not applicable for inorganic UVCB
IUPAC Name:
Slags, sponge iron production by coal reduction in retort
Test material form:
solid

Test animals

Species:
other: Human Epidermis Model EpiDerm™
Details on test animals or test system and environmental conditions:
Source - EpiDerm™ Reconstructed Human Epidermis Model Kit - supplier MatTek. Upon receipt of the tissues the sealed 24-well plate was placed into a refridgerator.

Test system

Amount / concentration applied:
Test material used as supplied
Duration of treatment / exposure:
3 minutes & 60 minute exposure periods.
Details on study design:
Pre-Test Procedure - Assessment of direct test item reduction of MTT

-50mg of test item wsa added to 900µL of a freshly prepared 1.0mg/mL MTT solution.
- The solution was incubated in the dark at 37°C, 5% CO2 in air for 3-hours
- 100µL of sterile distilled water was tested concurrently to act as a control
- The test item was found to have the ability to directly reduce MTT.

Main Test - Preincubabtion

- Assay medium was pre-warmed before use.
- 0.9mL of assay medium was pipetted into well of two pre-labelled 6-well plates for the 3 minutes and 60 minute exposure periods.
- The EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium and pre-incubated for approximately 1 hour before dosing at 37°C 5% CO2

Application of Test Item and Rinsing of test item

- After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9mL of fresh assay medium
- For the 60 minute exposure 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of
the test item and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 μL of sterile water was added for wetting of the test item. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.
- The procedure was repeated for the 3-minute exposure period.
- Rinsing was done by filling and emptying each tissue under a constant stream of DPBS to remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.
- Each tissue was transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2)) for 3 hours.
- Once the 60-minute exposure period was complete the same rinsing and MTT loading procedure was repeated.
- AFter the 3-hour MTT incubation period, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction.
- 2mL of MTT extractant (isopropanol) was used to completely immerse each insert and a sealed to prevent isopropanol evaporation and the plates were placed into a refridgerator overnight to allow extraction to proceed.

Absorbance/Optical Density Measurements

- After extraction each tissue was pierced with a pipette fittled with a 1000µL tip
- The extraction solution was mixed to form a homogenous solution
- Three 200µL aliquots of blue formazan extract was transferred to the pre-labelled plate
- 200µL L of isopropanol was added to the three well designated as blanks.
- Absorbency at 562nm (OD562) was measured using the Anthos 2001 microplate reader

Interpretation of Results
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 1000

Classification of the corrosivity potential is based on relative viabilities for both exposure times accoding to the attached table.

Quality Criteria : The results of the assay are considered acceptable if the following critera are acheived

Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is ≥ 0.8.
Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤ 30% relative to the negative control treated tissues following the 3-Minute exposure period




Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
83.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
95.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The relative mean viability of the test item treated tissues was 60 minute exposure 83.9% and 3 minute exposure 95.8%

Any other information on results incl. tables

Direct MTT Reduction

An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed no degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

Test Item, Positive Control Item and Negative Control Item

Mean OD562 values and viabilities for the negative control, positive control and test item are given in the attached table.

The relative mean viability of the test item treated tissues was as follows:

60 minute exposure : 83.9%

3 minute exposure : 95.8%

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 7.4% relative to the negative control treated tissues following the 3-Minute exposure period. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 2.127 for the 3-Minute exposure period and 2.304 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The corrosivity potential of the test item was assessed in accordance with OECD Guideline 431. The test item was considered to be non-corrosive to the skin.
Executive summary:

Summary

The study was performed to evaluate the corrositivity of the test item using the EpiDermTMSkin Model after treatment periods of 3 & 60 minutes

Results

Duplicate tissues treated with the test item for exposure periods of 3 and 60 minutes produced relative mean viabilities of 95.8 & 83.9% respectively.

Conclusion

The test item was considered to be non-corrosive to the skin

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