Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 25 June 2015 and 27 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:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on REACH
Deviations:
yes
Remarks:
See below
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid

Test animals

Species:
other: Reconstructed Human Epidermis
Strain:
other: Not applicable
Details on test animals and environmental conditions:
Supplier: MatTekDate received: 23 June 2015EpiDermTM Tissues (0.5cm2) lot number: 21677Assay Medium lot number: 0618152SBUpon receipt of the Epiderm tissues, the sealed 24-well plate was placed into a refrigerator.

Test system

Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
50 µL
Duration of treatment / exposure:
3 Minute and 60 Minute exposure periods
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
Pre-Test ProcedureAssessment of Direct Test Item Reduction of MTTMTT Dye Metabolism, Cell Viability AssayThe MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.Test for Direct MTT ReductionAs specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:100 µL of the test item was added to 900 µL of a 1.0 mg/mL MTT solution freshly prepared as above. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. 100 µL of sterile distilled water was tested concurrently to act as a control.If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.Main TestPre-IncubationThe assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.Application of Test Item and RinsingBefore pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 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. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and 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 was complete, the inserts were blotted and transferred to labeled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent isopropanol evaporation. The plates were placed into a refrigerator overnight, to allow extraction to proceed.Absorbance/Optical Density MeasurementsAfter extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: Relative Mean Viability
Value:
10.2
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 minutes. (migrated information)
Irritation / corrosion parameter:
other: other: Relative Mean Viability
Value:
58.7
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 minutes. (migrated information)

In vivo

Irritant / corrosive response data:
Direct MTT ReductionAn 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 corrosion 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 ItemThe relative mean viabilities of the test item treated tissues were as follows:60 minute exposure: 10.2%3 minute exposure: 58.7%

Any other information on results incl. tables

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 7.6% relative to the negative control treated tissues following the 3‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

The mean OD562for the negative control treated tissues was 2.153 for the 3‑Minute exposure period and 2.187 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied.

Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

Exposure Time

Mean OD5621

Percentage Viability

Negative Control

3 minutes

2.153

100*

60 minutes

2.187

100*

Positive Control

3 minutes

0.164

7.62

60 minutes

0.063

2.93

Test Item

3 minutes

1.263

58.72

60 minutes

0.223

10.23

*=   The mean viability of the negative control tissues is set at 100%

1=   Mean of EpiDermTMtissues tested in duplicate

2=   Viability expressed as a percentage of the 3 minute negative control tissues

3=   Viability expressed as a percentage of the 60 minute negative control tissues

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be corrosive to the skin.EU CLP (1272/2008/EC) and UN GHS Hazard statement H314 “Causes severe skin burns and eye damage” Category 1B.
Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

 

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

 

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 562 nm (OD562).

 

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Result

The relative mean viabilities of the test item treated tissues were:

 

60 minute exposure

:

10.2%

3 minute exposure

:

58.7%

 

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

 

Conclusion

The test item was considered to be corrosive to the skin.EU CLP (1272/2008/EC) and UN GHS Hazard statement H314 “Causes severe skin burns and eye damage” Category 1B