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:
28APR2015 - 01MAY2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted 26 September 2014
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
White powder with lumps and flakes
Details on test material:
- Colour: White

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot No.of test material: ZMG-187261
- Expiration date of the lot: 16 April 2016
- Purity test date: 31-07-2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: negative

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Normal, human-derived epidermal keratinocytes cultured to form a multilayered, highly differentiated model of the human epidermis
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure (3 minutes): room temperature
- Temperature used during exposure (1 hour) and during post-treatment incubation: 36.4 - 36.7°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing: not described
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test substance considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test substance is decreased below 15%.
- The test substance is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
- 26.0 to 26.7 mg test substance (the skin was moistened with 25 μl Milli-Q water to ensure close contact)
- 50 μl Milli-Q water (negative control)
- 50 μl 8N KOH (positive control)
Duration of treatment / exposure:
3 minutes or 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2/ incubation time

Test animals

Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.4 - 36.7
- Humidity (%): 75 - 87

Test system

Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 26.0 to 26.7 mg (skin tissue was moistened with 25 μl Milli-Q water to ensure close contact)

VEHICLE
The test substance was \moistened with \ mL of the vehicle.
Duration of treatment / exposure:
3-minute and 1-hour exposure
Number of animals:
2 tissues/ exposure period
Details on study design:
TEST SITE
- EpiDerm Skin Model (EPI-200, Lot no.: 22226 kit N; obtained from MatTek Corporation, Ashland MA, U.S.A). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 3 minutes or one hour

POST INCUBATION PERIOD
- 3 hours

SCORING SYSTEM:
- Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

CONTROLS:
Negative control: 50μL Milli-Q water (Millipore Corp., Bedford, Mass., USA).
Positive control: 50μL 8N KOH.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minutes incubation
Run / experiment:
1
Value:
86
Vehicle controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
14%
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour incubation
Run / experiment:
1
Value:
83
Vehicle controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
7%
Other effects / acceptance of results:
The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with MTBAC compared to the negative control tissues was 86% and 83% respectively. Because the mean relative tissue viability for MTBAC was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment MTBAC is considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 14% and 7% after 1 hour exposure. The maximum inter-tissue variability in viability between two tissues treated identically was less than 22% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%. It was therefore concluded that the test system functioned properly.

Any other information on results incl. tables

MTBAC was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that MTBAC did not interfere with the MTT endpoint.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
An in vitro skin corrosion test was conducted according to OECD 431 guideline and GLP principles. It is concluded that this test is valid and that MTBAC is not corrosive in the in vitro skin corrosion test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EpiDerm Skin Model), the influence of MTBAC on the viability of human skin was tested. The test substance was applied directly to 0.6 cm2 cultured skin (26.0 to 26.7 mg, in presence of 25 μl Milli-Q water). After 3 minutes or one hour, the substance was removed and cells were cultured for 3 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 14/% following 3 minute exposure whereas the test substance showed cell viability of 86%. Exposure for 60 minutes resulted in a mean cell viability of 83% and 7% for resp. MTBAC and the positive control. Since the mean relative tissue viability after exposure to MTBAC was above 50% after 3 minute exposure and above 15% after 1 hour exposure, it can be concluded that MTBAC is not corrosive in the in vitro skin corrosion test.