Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 December 2008 - 5 December 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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:
other: liquid (not specified)
Details on test material:
Description: dark brown liquid
Storage conditions: room temperature in the dark

Test animals

Species:
other: Reconstructed human epidermis model
Strain:
other: human-derived epidermal keratinocytes
Details on test animals and environmental conditions:
The EPISKIN model is a three-dimensional reconstituted 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 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKIN Model Kit
Supplier: SkinEthic Laboratories, Nice, France

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
50 µL.
Duration of treatment / exposure:
Exposure periods of 3, 60 and 240 minutes.
Observation period:
Not applicable.
Number of animals:
Not applicable.
Details on study design:
PRE-TEST
Assessment of Direct Test Material Reduction of MTT

MTT Dye Metabolism, Cell Viability Assay:
The MTT assay, a colourimetric 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 purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.

Assessment of Direct Test Material Reduction of MTT
One limitation of the assay is possible interference of the test material with MTT. A test materialmay directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test material 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 Reduction:
The test material was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test material was added to 2.2 mL of a 0.3 mg/mL MTT solution (v/v) freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

PRE-INCUBATION (Day 0: tissue arrival):
2.2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into two wells of the first column of a pre-labelled 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 material, control and time point. The tissues were incubated at 37 °C, 5 % CO2 in air for at least 24 hours.

MAIN TEST

APPLICATION OF TEST MATERIAL AND RINSING (Day 1)
2.2 mL of assay medium, warmed to approximately 37 °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 material 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 material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 µL of 0.9 % w/v sodium chloride solution was added for wetting of the test material. Duplicate tissues, treated with 50 µL of 0.9 % w/v 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 approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.

2.2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 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 room temperature in a biological safety cabinet ensuring that the plates were protected from light. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability).
Following qualitative evaluation of tissue viability, a total biopsy of the epidermis was taken using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 850 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 2)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.

For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as 'blanks'. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

INTERPRETATION OF RESULTS
Quantitative MTT Assessment (percentage tissue viability)

The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-minute treatments, 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 viability (%) = (mean OD540 of test material / mean OD540 of negative control) x 100

Classification of corrosivity potential was based on relative viabilities for each exposure time according to the prediction model:

Treatment Time Relative Mean Tissue Viability Prediction EU Risk Phrase UN Packing Group
(minutes) (percentage of negative control)
3 <35 Corrosive R35 I
3/60 ≥35 / <35 Corrosive R34 II
60/240 ≥35 / <35 Corrosive R34 III
240 ≥35 Non-Corrosive No label Non-Corrosive


QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
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.

Results and discussion

In vivo

Irritant / corrosive response data:
Test Material, Positive Control Material and Negative Control Material:
Individual and Mean OD540 Values and Viabilities for the Negative Control, Positive Control Material and Test Material are given in Table 1.

The relative mean viability of the test material treated tissues was as follows:
240 minutes exposure: 17.2 %
60 minutes exposure: 76.3 %
3 minutes exposure: 107.7 %

The qualitative evaluation of tissue viability is given in Table 2.

Following the 3 and 60-minutes exposure periods the test material treated tissues appeared blue which was considered to be indicative of viable tissue. Following the 240-minute exposure period the test material treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue.
Other effects:
Direct MTT Reduction
The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

Quality Criteria
The relative mean tissue viability for the positive control treated tissues was 4.1 % relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1 Individual and Mean OD540 Values and Viabilities

Material

Exposure Period (minutes)

Mean OD540 of Duplicate Tissues

Relative Mean % Viability

Negative control

240

0.169

100*

Positive control

240

0.007

4.1

 

Test material

240

0.029

17.2

60

0.129

76.3

3

0.182

107.7

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

 

Table 2 Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment)

Material

Exposure Period (minutes)

Tissue 1

Tissue 2

Negative control

240

-

-

Positive control

240

++

++

 

Test material

240

+

+

60

-

-

3

-

-

- = Blue tissue (viable)

+ = Blue/white tissue (semi-viable)

++ = Tissue completely white (dead)

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to have the potential to be corrosive to the skin and accredited the EU risk phrase R34 and UN packing group III.
Executive summary:

The potential corrosivity of the test material was investigated in vitro in accordance with the standardised guideline OECD 431 using the EPISKIN reconstituted human epidermis model. Tissues were treated with the test material for periods of 3, 60 and 240 minutes and the relative mean viability of the tissues assessed.

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

240 minutes exposure: 17.2 %

60 minutes exposure: 76.3 %

3 minutes exposure: 107.7 %

The test material was considered to have the potential to be corrosive to the skin and accredited the EU risk phrase R34 and UN packing group III.