Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 - 31 Aug 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
(adopted 13 April 2004)
Deviations:
yes
Remarks:
additional exposure period of 240 min; limit of evaluation criteria at 35% instead of 50%
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene diformate
EC Number:
211-077-7
EC Name:
Ethylene diformate
Cas Number:
629-15-2
Molecular formula:
C4H6O4
IUPAC Name:
ethylene diformate
Details on test material:
- Name of test material (as cited in study report): Ethylene Glycol Diformate
- Physical state: extremely pale yellow liquid
- Analytical purity: > 80% (w/w)
- Lot/batch No.: 13082
- Storage conditions: room temperature, in the dark

Test animals

Species:
human
Strain:
other: EpiSkin(TM); reconstructed three-dimensional human epidermis
Details on test animals or test system and environmental conditions:
TEST SKIN MODEL
- Source: SkinEthic Laboratories, Nice, France

TEST METHOD
The EPISKIN(TM) 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 (EPISKIN(TM) Model Kit 0.38 cm²). A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, spinous and granular layers and a functional stratum corneum. The procedure followed is based on the recommended EpiSkin(TM) Skin Corrosivity Test protocol INVITTOX No. 118. The test item is applied topically to the straturm 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 EPISKIN(TM) 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.

ADAPTATION TO CELL CULTURE CONDITIONS
Tissues were transferred into 12-well plates containing 2.2 mL of prewarmed maintenance medium and incubated for 2 days at 37 °C and 5% CO2.

Test system

Type of coverage:
open
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Controls:
other: concurrent control tissues treated with 0.9% w/v sodium chloride solution served as negative controls, positive controls were exposed to glacial acetic acid.
Amount / concentration applied:
TEST MATERIAL: 50 µL

NEGATIVE CONTROL SUBSTANCE: 0.9% (w/v) sodium chloride solution; 50 µL

POSITIVE CONTROL SUBSTANCE: glacial acetic acid; 50 µL
Duration of treatment / exposure:
Test item: 3, 60, and 240 min
Controls: 240 min
Observation period:
Not applicable
Number of animals:
Not applicable
The test was performed in duplicates for each test or control group and treatment period
Details on study design:
TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with PBS.
- Time after start of exposure: 3, 60 and 240 min

CELL VIABILITY MEASUREMENTS
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 filled wells. The tissues were incubated for 3 hours 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. A total biopsy of the epidermis was taken using the EPISKIN(TM) biopsy punch. The epidermis was carefully separated from the collagen matrix and both parts were plated 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.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured (quantitative viability analysis) at 540 nm using an Anthos 2001 microplate reader.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
100
Remarks on result:
other:
Remarks:
Basis: other: mean value of negative controls. Time point: 240 min. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
92.4
Remarks on result:
other:
Remarks:
Basis: other: mean value of the test item. Time point: 3 min. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
48.4
Remarks on result:
other:
Remarks:
Basis: other: mean value of the test item. Time point: 60 min. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
19.7
Remarks on result:
other:
Remarks:
Basis: other: mean value of the test item. Time point: 240 min. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
5.7
Remarks on result:
other:
Remarks:
Basis: other: mean value of positive controls. Time point: 240 min. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:
As the mean cell viability of the test item treated tissues was 19.7% compared to the negative control, the test item is considered as corrosive according to the evaluation criteria of this test.

Any other information on results incl. tables

The relative mean tissue viabilty for the positve control was 5.7% relative to the negative control. The mean OD540 for the negative control was 0.157. Thus, the acceptance criteria were satisfied.

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Category 1B Criteria used for interpretation of results: EU
Conclusions:
CLP: Skin Corrosive Cat. 1B, H314
DSD: C, R34