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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 October 2015 - 19 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Batch No: E00974-38D&F
Retest Date: 22 June 2018
Purity: 100%
Physical Description: Pale amber waxy solid
Storage Conditions: Ambient / dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The human keratinocytes came from healthy consenting donors
Justification for test system used:
The EpiSkin® in vitro corrosion test uses EpiSkin® tissues supplied by SkinEthic. The Episkin® reconstructed human epidermis model has been accepted as a valid model for skin corrosion assessment by the OECD.
Vehicle:
unchanged (no vehicle)
Details on test system:
The human keratinocytes came from healthy consenting donors. HIV 1 & 2, HEP B and HEP C tests were carried out on the donors as well as verification of the bacteriological and fungal sterility of the cells and absence of mycoplasma. After a period of culture (13 days) a
well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum were detectable. The surface area was 0.38 cm2.

The reproducibility of each batch was checked by histological analysis taking into account the general organisation, structure, and the reproducibility of the response of each EpiSkin batch was tested against a reference irritant: SDS.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
20 mg ± 2 mg
Duration of treatment / exposure:
3 min, 1 h +/- 5 min, or 4 h +/- 10 min
Number of replicates:
3 replicates per exposure time

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 mins
Value:
ca. 75.52
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour
Value:
ca. 118.44
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
4 hours
Value:
ca. 102.55
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The Substance did not reduce MTT.

The Substance did not become coloured upon mixing with ultrapure water.

Any other information on results incl. tables

See attached background documents

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Substance was demonstrated to be non-corrosive when tested in the EpiSkin in vitrocorrosion assay.
Executive summary:

In this study the corrosion potential of Substance was evaluated using the EpiSkin®in vitrocorrosion test.

 

Prior to the conduct of the corrosion assay, two preliminary tests were conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan, a measure of cell viability in the assay, and to assess the potential of the test item to cause colour interference. Substance did not reduce MTT to formazan or have the potential to cause colour interference, therefore, would not compromise the assay.

 

To assess dermal corrosion Substance was applied (20 mg ± 2 mg) to the exposed surface of EpiSkin®tissues for exposure periods of 4 h, 1 h and 3 min (3 tissues per exposure time). Due to the waxy nature of the test item it was heated toca 75°C then pipetted onto small filter papers, flattened onto the paper with a spatula and weighed. Filter papers were carefully placed on the exposed surface of the EpiSkin®tissue. The EpiSkin®surface area was 0.38 cm2, therefore the application rate wasca 52.6 mg/cm2. At the end of the exposure period, the filter papers were carefully removed and the surface of the Episkin®washed using Dulbecco’s phosphate-buffered saline (DPBS). The Episkin®tissues were transferred to MTT solution (0.3 mg/mL in assay medium) and incubatedin a humidified incubator set to maintain atemperature of 37°C and a CO2level of 5% for 3 h. Biopsies of the Episkin®membranes were removed, added to acidified isopropanol and left overnight, protected from light in order to extract formazan. The formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 570 nm.

 

Three replicates of each control (physiological saline, negative control, and acetic acid, positive control) were tested in parallel to test item treated tissues. Positive control was tested with the 4 h exposure group only, negative control was tested for all 3 exposure periods. The viability of each individual EpiSkinÒtissue was calculated as a percentage of the appropriate mean negative control viability (defined as 100%).

 

The mean A570of all negative control treated tissues was within 0.6-1.5 and the mean viability of positive control treated tissues was <20% therefore satisfying the assay acceptance criteria. Mean tissue viability (± SD) for test item treated tissues was 102.55 ± 16.53% (4h exposure), 118.44 ± 6.43% (1h exposure), and 75.52 ± 7.31% (3 min exposure).

 

In conclusion, Substance was demonstrated to be non-corrosive when tested in the EpiSkin in vitrocorrosion assay.