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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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 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 (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: clear colourless

In vitro test system

Test system:
human skin model
Remarks:
EPISKIN TM Small Model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiSkin RHE Model (Lot no.: 15-EKIN-050). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post exposure incubation period was also determined, as a complimentary endpoint.
The EPISKIN model is a three-dimensional reconstructed 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. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert. 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.
Application of test item and rinsing:
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
The tissues were subsequently placed into was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. Following 42 hour post exposure incubation the treated plates were then tested for MTT formazan extraction.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): undiluted
Duration of treatment / exposure:
Tissues were treated with the test material for 15 minutes and then washed with DPBS with Ca++ and Mg++ to remove residual test material.
Positive control: SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group.
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37°C.
Number of replicates:
Triplicate; treatment and concurrent negative control and positive control groups

Test system

Details on study design:
TEST SITE
- Area of exposure: 10 μl of the undiluted test substance was added topically into 12-well plates on top of the skin tissues. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean tissue viability
Value:
15.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minute exposure. Reversibility: no data. Remarks: n=3; SD = 3.3% ; Score in terms of percentage of negative control. (migrated information)
Irritation / corrosion parameter:
other: other: inflammatory mediator IL 1α release
Value:
225.677
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes exposure. Reversibility: no data. Remarks: n = 3; SD = 48.491; Mean Concentration (pg/mL). (migrated information)
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data (n=50): the mean OD of the positive control was 0.103; range 0.049 to 0.243 and the mean percentage viability was 12.2%; range 4.0% to 29.4%. In this same period the mean OD of the negative control was 0.854; range 0.629 – 1.245. The mean OD562 for the negative control treated tissues was imperceptibly outside the lower limit. However, this was within the OECD TG 439 guideline acceptance range and all other values obtained within the test fell within the historical range, therefore the test system performed adequately.

Any other information on results incl. tables

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

Item

OD562of tissues

Mean OD562 of triplicate tissues

±SDof OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.608

0.626

0.022

97.1

100*

3.5

0.650

103.8

0.619

98.9

Positive Control Item

0.081

0.089

0.014

12.9

14.2

2.2

0.082

13.1

0.105

16.8

Test Item

0.100

0.095

0.020

16.0

15.2

3.3

0.113

18.1

0.073

11.7

OD = Optical Density

SD = Standard deviation

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

 

The relative mean tissue viability for the positive control treated tissues was 14.2% relative to the negative control treated tissues and the standard deviation value of the viability was 2.2%. The mean OD562 for the negative control treated tissues was 0.626 and the standard deviation value of the viability was 3.5%. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 3.3%. All assay acceptance criteria were met.

 

Furthermore, within the Historic Control Data (HCD) (n=50): the mean OD of the positive control was 0.103; range 0.049 to 0.243 and the mean percentage viability was 12.2%; range 4.0% to 29.4%. In this same period the mean OD of the negative control was 0.854; range 0.629 – 1.245. The mean OD562 for the negative control treated tissues was imperceptibly outside the lower limit. However, this was within the OECD TG 439 guideline acceptance range and all other values obtained within the test fell within the historical range, therefore the test system performed adequately.

 

The mean concentration of inflammatory mediator IL 1α in the culture medium retained from the test item treated tissues was 225.677 pg/mL.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test material is considered to be irritating to skin.
Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test substance in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). After a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for inflammatory mediator IL-1α determination. 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 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 15.2% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The inflammatory mediator IL 1α in the retained culture medium was measured. The mean concentration of inflammatory mediator IL 1α in the culture medium retained from the test item treated tissues was 225.677 pg/mL. All assay acceptability criteria were met. Under the conditions of this study, the test substance is considered irritating to the skin and would be considered to meet the criteria under Regulation (EC) 1272/2008 for Skin Irritation Category 2: Irritant.