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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD guideline 431 (In vitro skin corrosion: human skin model test)
Principles of method if other than guideline:
The test material 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 sufficiently cytotoxic after a short term exposure to the
EPISKIN 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 material treated cultures relative to the negative control.
GLP compliance:
yes (incl. QA statement)
Remarks:
Harlan Laboratories Ltd. Shardlow Business Park Shardlow Derbyshire DE72 2GD UK

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyloxirane
EC Number:
200-879-2
EC Name:
Methyloxirane
Cas Number:
75-56-9
Molecular formula:
C3H6O
IUPAC Name:
1,2-Epoxypropane
Details on test material:
Sponsor's identification : Propylene oxide
Description : clear colourless liquid
Batch number : S-122009-310041
Date received : 14 December 2009
Expiry date: 14 June 2010
Storage conditions : room temperature in the dark
Purity:99.98 %

Test animals

Species:
other: The EPISKIN model is a three-dimensional reconstituted human epidermis modelconsisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type lV collagen.
Strain:
not specified
Details on test animals or test system and environmental conditions:
The EPISKINTM model is a three-dimensional reconstituted human epidermis modelconsisting of adult human-derived epidermal keratinocytes
seeded on a dermal substitute consisting of a collagen type I matrix coated with type lV 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.

EPISKN ModeI Kit
Supplier : SkinEthic Laboratories, Nice, France
Date received : 26 January 2010

Test system

Type of coverage:
open
Preparation of test site:
other: use of the Episkin model kit
Vehicle:
not specified
Controls:
not required
Amount / concentration applied:
50 microliter of the test material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues.
Duplicate tissues, treated with 50 microliter of 0.9% w/v sodium chloride solution served as negative controls.
Duplicate tissues, treated with 50 microliter of glacial acetic acid served as positive controls.
Duration of treatment / exposure:
3, 60 and 240 minutes
Observation period:
not required
Number of animals:
not required
Details on study design:
The MTT assay, a colourimetríc method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide) to a purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.

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 materials for an exposure period of 240 rninutes. 50 microliter of the test material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. Duplicate tissues, treated with 50 microliter of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 microliter of glacial acetic acid served as positive controls. The treated tissues were kept in the 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 wererinsed.

2.2 ml of 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 microliter 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.

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 microliter samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 microliter 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 fifter) using the Anthos 2001 microplate reader.









Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: relative mean tissue viability
Value:
96.4
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 240 minutes. (migrated information)
Irritation / corrosion parameter:
other: other: relative mean tissue viability
Value:
119.4
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 minutes. (migrated information)
Irritation / corrosion parameter:
other: other: relative mean tissue viability
Value:
141.3
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 minutes. (migrated information)
Irritation / corrosion parameter:
other: other: relative mean tissue viabilty
Value:
5.6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 240 minutes. Remarks: positive control. (migrated information)

Any other information on results incl. tables

The test material was considered to be Non-Corrosive to the skin.

Applicant's summary and conclusion