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:
key study
Study period:
March 17-19, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 431 (In vitro skin corrosion: Human skin model test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): FLL sample 4
- Physical state: viscous liquid
- Lot/batch No.: 240210
- Expiration date of the lot/batch: not reported
- Storage condition of test material: room temperature in the dark
- Other: yellow/brown color
- All other template details: Not reported

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Human-derived epidermal keratinocytes seeded on a collagen type I matrix coated with type IV collagen.
Cell source:
other: Reconstituted model.
Source strain:
not specified
Details on animal used as source of test system:
Not applicable.

- EPISKIN Model Kit
- Source: SkinEthic Laboratories, Nice, France
- Date received: 16 March 2010
Justification for test system used:
The EPISKIN model is scientifically validated for use as replacement for the animal test.
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: (Preincubation): 2.2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test material, control and time point. The tissues were incubated at 37°C, 5% CO2 in air for at least 24 hours. After 24 hours the media under the tissues was refreshed.
- Quality control for skin discs: Not specified.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature.
- Temperature of post-treatment incubation (if applicable): Room temperature.
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: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..
- Observable damage in the tissue due to washing: None reported.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: The optical density was measured at 540 nm without a reference filter.

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Duplicate tissues were treated with the test item.
PREDICTION MODEL / DECISION CRITERIA
Classification of corrosivity potential was based on relative viabilities for each exposure time according to the prediction model of the table 1.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL (undiluted).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL.
- Concentration (if solution): 0.9 % (w/w) sodium chlorite solution.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL.
- Concentration (if solution): Glacial acetic acid.
Duration of treatment / exposure:
3, 60 and 240 minutes
Duration of post-treatment incubation (if applicable):
3 hours with MTT solution.
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item / 3 minutes
Value:
108.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item/ 60 minutes
Value:
99.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item/ 240 minutes.
Value:
105
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative control / 240 minutes
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control / 240 minutes
Value:
9.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

In vivo

Irritant / corrosive response data:
See Table 1 for raw data values
Other effects:
Test material was shown to directly reduce MTT. However, rinsing was effective at removing the test material on or in the tissue following each exposure period and no degree of interference was noted.

Any other information on results incl. tables

Table 1. Individual and Mean OD540 Values and Viabilities for the Negative Control Material, Positive Control Material and Test Material.

Material

Exposure Period (minutes)

OD540 of duplicate tissues

Mean OD540 of duplicate tissues

Relative mean viability (%)

Negative control material

240

0.150

0.161

100

0.172

Positive control material

240

0.013

0.015

9.3

0.016

Test material

240

0.187

0.169

105.0

0.150

60

0.172

0.160

99.4

0.148

3

0.173

0.174

108.1

0.174

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
CLP implementation.
Conclusions:
The test material was considered to be non-corrosive to the skin.
Executive summary:

Summary from data report:

Introduction: The purpose of this test is to evaluate the corrosivity potential of the test material using the EPISKINTMin vitro Reconstituted Human Epidermis (RHE) Model after treatment periods of 3, 60, and 240 minutes. This method was designed to meet the requirements of the OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

The EPISKINTMmodel is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group 1) and R34 (UN packing group II&III) chemicals.

 

Methods: Duplicate tissues were treated with the test material for exposure periods of 3, 60, and 240 minutes. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues.

 At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

 Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to the negative control tissues).

 

Results: The relative mean viability of the test material treated tissues was:

 240 minutes exposure:    105.0%

 60 minutes exposure:      99.4%

 3 minutes exposure:      108.1%

 Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

 

Conclusion: The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.