Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 January 2013 to 29 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid (unspecified)
Details on test material:
- Appearance: red-brown liquid
- Storage conditions: room temperature in the dark
- Stability under storage conditions: stable

Test animals

Species:
other: in vitro Reconstructed Human Epidermis (RHE) Model
Details on test animals and environmental conditions:
The EPISKIN model is a three-dimensional 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. The keratinocytes were cultured for 13 days, resulting in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKIN Small Model Kit 0.38cm²
Supplier: SkinEthic Laboratories, Lyon, France
Batch number: 13-EKIN-002

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro positive and negative controls were used
Amount / concentration applied:
25 μL
Duration of treatment / exposure:
A treatment period of 15 minutes was followed by a post-exposure incubation period of 42 hours.
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
MATERIAL PREPARATION
- Phosphate buffered saline (PBS) was used as the negative control.
- 5 % (aqueous) Sodium dodecyl sulphate (SDS) in PBS was used as the positive control.

TEST SYSTEM
PREPARATION AND PRE-INCUBATION
On the day of receipt, the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 6 hours and 18 minutes at 37 °C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories.

MTT medium:
MTT concentrate (3 mg/mL in PBS) diluted (10 x) in Assay medium (final concentration 0.3 mg/mL).

Environmental conditions:
All incubations, with the exception of the test material incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 % (actual range 76 to 98 %), containing 5.0 ± 0.5 % CO₂ in air in the dark at 37.0 ± 1.0 °C (actual range 36.6 to 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 4 %) 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.

TEST FOR REDUCTION OF MTT BY THE TEST MATERIAL
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, 25 μL of the test material was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3.5 hours at 37 °C. A negative control, sterile Milli-Q water was tested concurrently.
No colour change was observed and it was concluded that the test material did not reduce the MTT directly.

APPLICATION/TREATMENT OF THE TEST MATERIAL
The test was performed on a total of 3 tissues per test material together with negative and positive controls. 25 μL of the undiluted test material was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS and 3 tissues with 25 μL 5 % SDS. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test material. 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.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 hours at 37 °C. After incubation, the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL of isopropanol. Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite M200 Pro Plate Reader (Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement).
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test material was classified according to remaining cell viability following exposure of the test material.

INTERPRETATION
Acceptability of the assay:
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
- The mean relative tissue viability of the positive control should be ≤40 % relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
- The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Data evaluation and statistical procedures:
A test material is considered irritant in the skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is ≤50 % of the mean viability of the negative controls.

A test material is considered non-irritant in the in vitro skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is >50 % of the mean viability of the negative controls.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
113
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
24% tissue viability after 15 minutes exposure
Remarks on result:
no indication of irritation

Any other information on results incl. tables

The positive control had a mean cell viability after 15 minutes exposure of 24 %. The absolute mean OD570 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 7 %, indicating that the test system functioned properly.

 

Table 1: Mean Absorption Values

Material

OD540 Values

Mean OD540 Values

Standard Deviation

A

B

C

Negative Control

0.940

0.857

0.983

0.927

0.064

Test Material

0.997

1.112

1.027

1.045

0.060

Positive Control

0.211

0.252

0.205

0.223

0.026

Triplicate exposures are indicated by A, B and C

Values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 

Table 2: Mean Tissue Viability

Material

Mean Tissue Viability

(% of control)

Negative Control

100

Test Material

113

Positive Control

24

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test material was determined to be non-irritating and requires no classification in accordance with EU criteria.
Executive summary:

The skin irritation potential of the test material was evaluated in vitro using the EPISKIN reconstructed human epidermis model in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period, each tissue was rinsed before incubating for 42 hours after which each tissue was taken for MTT loading. After MTT loading, a total biopsy of each epidermis was made and formazan crystals were extracted out of the MTT-loaded tissues. Duplicate tissues treated with PBS served as the negative control and duplicate tissues treated with 5 % aqueous Sodium Dodecyl Sulphate in PBS served as the positive control. The optical density of all treated tissues was measured at 540 nm.

The relative mean viability of the test material treated tissues was 113 % after the 15 minute exposure period.

Under the conditions of this study, the test material was determined to be non-irritating and requires no classification in accordance with EU criteria.