Registration Dossier

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: The study was conducted according to GLP, OECD TG 439 and other similar study guidelines without deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Principles of method if other than guideline:
Protocol deviations: The 12 wells plates with tissues were preincubated for 2 hours instead of 24 hours.
Evaluation: Shipment of the tissue kit took longer than expected. After consultation with the
supplier the advice was to transfer the Episkin tissues to maintenance medium for at least
2 hours instead of 24 hours. Since all acceptability criteria were met this has no adverse effect
on the study integrity.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification
TRANTOS

Molecular formula
C26H36O4S
Molecular weight
444.63
CAS Number
10429-07-9
Description
White powder (determined at WIL Research Europe B.V.)
Batch
CQE299K1DL2
Purity/composition
99.9%
Test substance storage
At room temperature in the dark
Stability under storage conditions
Stable
Expiry date
15 January 2014 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)

Test animals

Species:
other: in vitro: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-009,
Details on test animals and environmental conditions:
Test system
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-009, See APPENDIX 4).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Source
SkinEthic Laboratories, Lyon, France.

Test system

Type of coverage:
open
Preparation of test site:
other: in vitro
Controls:
yes
Amount / concentration applied:
The solid test substance (12.7 to 14.2 mg) was applied directly on top of the skin tissue. TRANTOS was spread to match the size of the tissue.
Duration of treatment / exposure:
After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C
Observation period:
not applicable
Number of animals:
3 tissues per test substance together with negative and positive controls
Details on study design:
Tissues
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 2 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.


MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
Environmental conditions
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 - 97%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.9 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 20%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
103
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 100.0. Remarks: The relative mean tissue viability obtained after 15 minutes treatment with TRANTOS compared to the negative control tissues was 103%. Since the mean relative tissue viability for TRANTOS was above 50% TRANTOS is considered to be non-irritant.. (migrated information)

In vivo

Irritant / corrosive response data:
Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with TRANTOS compared to the negative control tissues was 103%. Since the mean relative tissue viability for TRANTOS was above 50% TRANTOS is considered to be non-irritant.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
TRANTOS is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

In vitro skin irritation test with TRANTOS using a human skin model. This report describes the ability of TRANTOS to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of TRANTOS was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch CQE299K1DL2 of TRANTOS was a white solid with a purity of 85-95%. Skin tissue was moistened with 10 μl of Milli-Q water and 12.7 to 14.2 mg of TRANTOS was applied directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with TRANTOS compared to the negative control tissues was 103%. Since the mean relative tissue viability for TRANTOS was above 50% after 15 minutes treatment TRANTOS is considered to be non-irritant. The positive control had a mean cell viability of 19% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 13%, indicating that the test system functioned properly. Finally, it is concluded that this test is valid and that TRANTOS is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.