Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
yes
Remarks:
Minor deviation in that the test tissues were stored refrigerated for 48 hours prior to testing; permitted under model SOP and guideline and which does not affect the reliability of the study
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
yes
Remarks:
Minor deviation; see above.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected June 2015; signature: September 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid.- Storage condition of test material: Approximately 4°C in the dark- Other: Pale yellow

In vitro test system

Test system:
human skin model
Source species:
human
Details on test system:
EpiDerm Skin Model (EPI-200-SCT, Lot no.: 21697). This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. Pre-test checks for Direct MTT reduction and Colour Interference were completed.Preincubation:The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.Application of test item and rinsing:After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. Tissues were then dosed at regular intervals with test item/negative control and/or positive control respectively for the appropriate exposure times. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. The plate was incubated (37 °C, 5% CO2) for 3 hours. After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction.

Test animals

Species:
other: EpiDerm Skin Model (EPI-200-SCT, Lot no.: 21697)
Strain:
not specified

Test system

Controls:
yes
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 50 μl- Concentration (if solution): undiluted
Duration of treatment / exposure:
Two tissues were used for a 3-minute exposure to the test substance and two for a 60-minute exposure.
Number of animals:
The test was performed on a total of 4 tissues per test substance (duplicate: 3-minutes and 6-minutes) together with a negative control and positive control in duplicate.
Details on study design:
TEST SITE- Area of exposure: 50 μl of the undiluted test substance was added on top of the skin tissues.REMOVAL OF TEST SUBSTANCE- Washing (if done): yes- Time after start of exposure: Two tissues were washed after 3 minutes and two tissues were washed after 60 minutes with Dulbeccos PBS (DPBS).SCORING SYSTEM: Skin corrosion is expressed as the remaining cell viability after exposure to the test substance at exposure times 3-minutes and 60-minutes, respectively. Where necessary, direct MTT reduction, colour interference and correction for background Isopropanol absorbance at OD562 (via measurement of triplicate blanks) was completed.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of two runs (n = 2); 3 minutes exposure to the test material
Value:
118.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indications of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of two runs (n = 2); 1 hour exposure to the test material
Value:
116.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indications of corrosion

In vivo

Irritant / corrosive response data:
Table 1 shows the mean tissue viability obtained after 3-minute and 60-minute treatments with test substance compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance.

Any other information on results incl. tables

Table 1. Mean absorption and tissue viability in the in vitro skin corrosion test with the test substance

Tissue

Exposure Period

Mean OD562 of individual tissues

Mean OD562 of duplicate tissues

Standard Deviation

Coefficient of Variation
(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.865

1.843

0.03

1.7

100*

1.821

60 Minutes

1.891

1.919

0.04

2.1

1.947

Positive Control

3 Minutes

0.116

0.114

0.003

N/A

6.2

0.112

60 Minutes

0.098

0.112

0.02

N/A

5.8

0.125

Test Item

3 Minutes

2.271

2.186

0.1

5.5

118.6

2.101

60 Minutes

2.270

2.241

0.04

1.8

116.8

2.212

* = The mean % viability of the negative control tissue is set at 100%

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the test substance is not considered to be corrosive in the in vitro skin corrosion test using EpiDerm Skin Model.
Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test substance in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). The mean OD562 for the negative control treated tissues was 1.843 for the 3 Minute exposure period and 1.919 for the 60 Minute exposure period. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 118.6% and 116.8% respectively. The relative mean tissue viability for the positive control treated tissues was 5.8% relative to the negative control following the 60 Minute exposure period. The quality criteria required for acceptance of results in the test were satisfied. Under the conditions of this study, the test item was considered to be non-corrosive to the skin.