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:
20-26 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study

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:
not specified
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
not specified
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: dark brown liquid
Details on test material:
Reaction Product of 1-chloro-2,3-epoxypropane, sodium hydroxide, sodium carbonate and water (2% sodium hydroxide) (Lot# 1H03192011) was used. Sodium hydroxide was added to provide 2% sodium hydroxide, the maximum in production material.

The test item was used as supplied.

Test animals

Species:
human
Strain:
not specified
Details on test animals and environmental conditions:
EPISKIN™ MODEL KIT was obtained from Skin Ethic Laboratories, Lyon, France.

Test system

Type of coverage:
open
Preparation of test site:
other:
Vehicle:
unchanged (no vehicle)
Controls:
other: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control. An aqueous solution of Sodium Dodecyl Sulphate (SDS) 5% w/v was used as the positive control.
Amount / concentration applied:
10 uL
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Number of animals:
Tests were run in triplicate
Details on study design:
MTT dye metabolism, cell viability assay
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:

10 uI of the test item was added to 2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT.

The test item was shown to directly reduce MTT in the direct MTT reduction test (section 8.1: Direct MTT Reduction). There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test substance like viable tissues. Water-killed tissues were prepared by placing untreated EPISKIN™ tissues in a 12-well plate containing 2.2 ml of sterile distilled water in each well. The tissues were incubated at 37°C, 5% CO2 in air for 48 ±1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30°C) for up to 6 months. Before use each tissue was thawed by placing in 2.2 ml of maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, each MTT reducing test substance was applied to a water-killed tissue. In addition, one water-killed tissue remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.

Pre-Incubation (Day 0: tissue arrival)
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.

Main Test
Application of Test Item and Rinsing (Day 1)
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 uI of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 uI of OPBS served as the negative controls and triplicate tissues treated with 10 uI of SOS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SOS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SOS solution was re-spread with a pipette tip to maintain the distribution of the SOS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing OPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of OPBS 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.

MTT Loading/Formazan Extraction (Day 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise 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°C for possible inflammatory mediator determination.

2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MIT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% C02 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 uI of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MIT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.

For each tissue, duplicate 200 uI samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 uI of acidified isopropanol alone was added to the two wells designated as 'blanks'. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: relative tissue viability
Value:
95
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 hour. Reversibility: no data. (migrated information)

In vivo

Irritant / corrosive response data:
Direct MTT Reduction
The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT.

The direct reduction by the test item relative to the negative control value was 6.2%

Direct reduction was therefore acceptable.

Test Item, Positive Control Item and Negative Control Item
The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test item treated tissues was 95.0% after a 15-Minute exposure period.
Other effects:
No additional information available

Any other information on results incl. tables

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.3%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.744 and the standard deviation value of the percentage viability was 11.6%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 3.1 %. The test item acceptance criterion was therefore satisfied.

Table 1

DETERMINATION OF SKIN IRRITATION POTENTIAL USING THE EPISKIN™ RECONSTRUCTED

HUMAN EPIDERMIS MODEL

Mean OD540 Values and Percentage Viabilities for Control Item, Positive Control Item and Test Item

 Material  OD540 of tissues tvt  OD540 of tissues corrected for direct reduction of MTT (0.046)  Mean OD540 (SD) Relative individual tissue viability (%)    Relative mean viability (%) (SD)
 Negative Control  0.751    0.744 (0.086)  100.9  100 (11.6)*
   0.655      88.0  
   0.827      111.2  
 Positive Control  0.045    0.062 (0.017)  6.0  8.3 (2.3)
   0.061      8.2  
   0.079      10.6  
 Test Item  0.727  0.681  0.707 (0.023)  91.5  95.0 (3.1)
   0.759  0.713    95.8  
   0.772  0.726    97.6  

Corrected viability of treated killed tissues = 0.130 (tkt) - 0.084 (ukt) = 0.046

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

OD540 = optical density

SD = Standard deviation

tvt = treated viable tissues

tkt = treated killed tissues

ukt = untreated killed tissues

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be Non-Irritant (NI).
Executive summary:

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis 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 colourimetric 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. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will

be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

• OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010)

• Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Method: 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 postexposure incubation period each tissue was taken for MTT-Ioading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator 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 540 nm.

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

Results: The relative mean viability of the test item treated tissues was 95.0% after the 15-Minute exposure period.

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

Conclusion: The test item was considered to be Non-Irritant (NI).