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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:
10 - 13 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with current OECD guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
No data on the test material referenced in the study report.

In vitro test system

Test system:
human skin model
Source species:
human

Test animals

Species:
other: Reconstructed Human Epidermis (RHE) Model
Strain:
not specified
Details on test animals and environmental conditions:
The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKINTM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.Skin corrosion refers to the production of irreversible tissue damage in the skin following theapplication of a test item (as defined by the Globally Harmonised System (GHS) for the Classification and Labelling of Chemical Substances and Mixtures). The potential for chemical induced skin corrosion is an important consideration in establishing procedures for the safehandling, packing and transport of chemicals. Validation studies have shown that testsemploying human skin models are able to reliably distinguish between known skin corrosivesand non-corrosives (Botham et al., 1995, Barrett et al., 1998 and Fentem et al., 1998). At its 10th Meeting, held on 31 March 1998 at ECVAM, Ispra, Italy, the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the EPISKINTM model as scientifically validated for use as a replacement for the animal test. The EPISKINTM model is able to distinguish betweencorrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.The procedure followed is based on the recommended EpiSkinTM Skin Corrosivity Test protocol INVITTOX No 118. The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKINTM model. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.

Test system

Type of coverage:
other: in vitro model
Preparation of test site:
other: in vitro model
Vehicle:
unchanged (no vehicle)
Remarks:
in vitro model
Controls:
not required
Amount / concentration applied:
50 µL of the test item was added to 2.0 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium.
Duration of treatment / exposure:
3, 60 and 240 minutes
Observation period:
3 days (see below)
Number of animals:
none - in vitro method
Details on study design:
Reference Items Information as provided by the suppliers.Identification:0.9% w/v sodium chloride solutionBatch:300999 104Purity:0.9%Expiry Date:01 January 2015Storage Conditions:room temperatureIdentification:Glacial acetic acidBatch:114957Purity:>99.7%Expiry Date:08 November 2014Storage Conditions:room temperatureMATERIALS AND METHODSTest SystemEPISKIN™ Reconstructed Human Epidermis Model KitSupplier : SkinEthic Laboratories, Lyon, FranceDate received : 10 September 2013EpiSkinTM Tissues (0.38cm2) lot number : 13-EKIN-031Maintenance Medium lot number : 13-MAIN3-038Assay Medium lot number : 13-ESSC-032Test Item Formulation and Experimental PreparationThe test item was used as supplied.Preparation of Negative and Positive Control ItemsThe negative control item was used as supplied. The positive control item was used as supplied.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 is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:50 µL of the test item was added to 2.0 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin corrosion potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.Pre-Incubation (Day 0: tissue arrival)Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:Tissues Satisfactory : Yes Temperature Indicator Color Satisfactory : Yes Agar Medium Color Satisfactory : Yes2.0 mL of maintenance medium, warmed to approximately 37 ºC, was pipetted into two wells of the first column of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test item, control and time point. The tissues were incubated at 37 deg C, 5% CO2 in air overnight. After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.Main TestApplication of Test Item and Rinsing (Day 2)2.0 mL of assay medium, warmed to approximately 37 deg C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 50 µL of the test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. Duplicate tissues, treated with 50 µL of 0.9% w/v Quality CriteriaThe results of the assay are considered acceptable if the following assay acceptance criteria are achieved:Negative Control:The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is ≥0.600 and ≤ 1.500.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 0 to 20% relative to the negative control treated tissues following the 240-Minute exposure period.sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µL of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.2.0 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours ±5 minutes at 37 deg C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.Absorbance/Optical Density Measurements (Day 3)At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.For each tissue, duplicate 200 L samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 L of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at562 nm (without a reference filter) using the Anthos 2001 microplate reader.Interpretation of ResultsQuantitative MTT Assessment (percentage tissue viability)The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloride solution. The relative mean viabilities were calculated in the following way:Relative mean visibility (%): (mean OD562 of test item / mean OD562 of negative control) x 100

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: Mean OD562 values
Remarks:
relative mean viabilities
Run / experiment:
3 minutes time point
Value:
114.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: Mean OD562 values
Remarks:
relative mean viabilities
Run / experiment:
60 minutes time point
Value:
107.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: Mean OD562 values
Remarks:
relative mean viabilities
Run / experiment:
240 minutes time point
Value:
120.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Direct MTT ReductionThe MTT solution containing the test item remained yellow. This was taken to indicate that the test item did not reduce MTT.Test Item, Positive Control Item and Negative Control ItemMean OD562 values and viabilities for the negative control, positive control and test item are given in Table 1 below.
Quality CriteriaThe relative mean tissue viability for the positive control treated tissues was 3.6% relative to the negative control treated tissues following the 240-Minute exposure period. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 0.868. The negative control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

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

240 minutes exposure

:

120.4%

60 minutes exposure

:

107.5%

3 minutes exposure

:

114.9%

Table1     Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item

Exposure Period

Mean OD562of individual tissues

Mean OD562of duplicate tissues

Relative mean viability (%)

Negative Control Item

240 Minutes

0.871

0.868

100*

0.865

Positive Control Item

240 Minutes

0.029

0.031

3.6

0.032

Test Item

240 Minutes

1.118

1.045

120.4

0.972

60 Minutes

0.925

0.933

107.5

0.941

3 Minutes

0.989

0.997

114.9

1.004

 

Å


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

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-corrosive to the skin. The following classification criteria apply:EU DSD (67/548/EEC): Not classified for corrosivity.EU CLP (1272/2008/EC)/UN GHS: Not classified for corrosivity. UN Packing Group: Non-Corrosive.
Executive summary:

The test item was classified as non-corrosive to the skin. The following classification criteria apply:

 EU DSD (67/548/EEC): Not classified for corrosivity.

 EU CLP (1272/2008/EC)/ UNGHS: Not classified for corrosivity.

UN Packing Group:Non-Corrosive.