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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28th to 30th April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted to GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: CAS No 10584-98-2
Description: clear colourless liquid
Batch number: 20100409995
Date received: 26 March 2010
Expiry date: 26 March 2012
Storage conditions: approximately 4°C in the dark under nitrogen

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Test animals

Species:
other: Reconstituted Human Epidermis (RHE)
Strain:
other: Not applicable
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 a 13 day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKIN Model Kit 0.38 cm2
Supplier: SkinEthic Laboratories, Nice, France
Date received: 27 April 2010

Test system

Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: 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.
Amount / concentration applied:
50 µl of the test material was applied
Duration of treatment / exposure:
Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes.
Observation period:
3, 60 and 240 minutes.
Number of animals:
Not applicable

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Relative mean viability (%)
Basis:
other: n/a
Time point:
other: 3 minutes
Score:
102.9
Reversibility:
other: n/a
Irritation parameter:
other: Relative mean viability (%)
Basis:
other: n/a
Time point:
other: 60 minutes
Score:
108.2
Reversibility:
other: n/a
Irritation parameter:
other: Relative mean viability (%)
Basis:
other: n/a
Time point:
other: 240 minutes
Score:
96.6
Reversibility:
other: n/a
Irritant / corrosive response data:
Direct MTT Reduction:
An assessment found the test material was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin corrosion potential. However, the results obtained showed that no degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Test Material, Positive Control Material and Negative Control Material:
Mean OD540 values and viabilities for the negative control, positive control and test material are given in Table 1.
The relative mean viability of the test material treated tissues was as follows:
240 minutes exposure: 96.6%
60 minutes exposure: 108.2%
3 minutes exposure: 102.9%

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

Following the 3, 60 and 240 Minute exposure periods the test material treated tissues appeared blue which was considered to be indicative of viable tissue.

Quality Criteria:
The relative mean tissue viability for the positive control treated tissues was 4.8% 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

The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.

Table 1:              Mean OD540Values and Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

Exposure Period

Mean OD540of duplicate tissues

Relative mean viability (%)

Negative Control Material

240 Minutes

0.207

100*

Positive Control Material

240 Minutes

0.010

4.8

Test Material

240 Minutes

0.200

96.6

60 Minutes

0.224

108.2

3 Minutes

0.213

102.9

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

Table 2              Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Exposure Period

Tissue 1

Tissue 2

Negative Control Material

240 Minutes

-

-

Positive Control Material

240 Minutes

++

++

Test Material

240 Minutes

-

-

60 Minutes

-

-

3 Minutes

-

-

MTT Visual Scoring Scheme of EpiSkin Tissues

- = Blue tissue (viable)

+ = Blue/white tissue (semi-viable)

++ = Tissue completely white (dead)

Applicant's summary and conclusion

Interpretation of results:
other: Non-Corrosive to the skin
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.
Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test material using the EPISKINTM in vitro Reconstituted Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to meet the requirements of the OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

The EPISKIN model is able to distinguish between corrosive 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) chemicals.

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density was measured at 540 nm.

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

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

240 minutes exposure: 96.6%

60 minutes exposure: 108.2%

3 minutes exposure: 102.9%

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

The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.