Reaction mass of 2-hexadecyl-N-(2-hydroxyethyl)-3-oxoicosanamide, 2-hexadecyl-N-(2-hydroxyethyl)-3-oxo-, N-(2-hydroxyethyl)-3-oxo-2-tetradecylicosanamide and N-(2-hydroxyethyl)-3-oxo-2-tetradecyloctadecanamide.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 30 August 2011 and 1st September 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Principles of method if other than guideline:

Validation studies have shown that tests employing human skin models are able to reliably distinguish between known skin corrosives and non-corrosives. At its 10th Meeting, held on 31 March 1998 at ECVAM, Ispra, Italy, the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the EPISKIN™ model as scientifically validated for use as a replacement for the animal test. The EPISKIN™ model 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 I) and R34 (UN packing group II & III) test items.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

PC-9S

IUPAC Name:

PC-9S

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: PC-9S
Description: off white powder
Lot number: PS101103
Purity: not supplied
Date received : 11 July 2011
Expiry date: not supplied
Storage conditions: room temperature in the dark

Test animals

Species:

human

Strain:

other: reconstructed human epidermis model

Details on test animals or test system and environmental conditions:

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 the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Supplier: SkinEthic Laboratories, Nice, France
Date received: 30 August 2011

Test system

Type of coverage:

other: not appliacble as an in vitro method was used

Preparation of test site:

other: not applicable in vitro method was used

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Application of Test Item and Rinsing (Day 1)
2.2 ml of assay medium, warmed to approximately 37°C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 20 mg of the solid test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 µI of 0.9% w/v sodium chloride solution was added for wetting of the test item. Duplicate tissues, treated with 50 µI of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µI of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.

Duration of treatment / exposure:

3, 60 and 240 minutes

Observation period:

At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. 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 item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.

The tissues were incubated for 3 hours ±5 minutes at room temperature in a biological safety cabinet ensuring that the plates were protected from light. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry.

Number of animals:

duplicate

Details on study design:

The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKIN™ model. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.

The following pre test was conducted:

As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below: 20 mg of the test item was added to 2.2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.

Pre-Incubation period:
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 item, control and time point. The tissues were incubated at 37°C, 5% CO2 in air overnight.

Main Test:
2.2 ml of assay medium, warmed to approximately 37°C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 20 mg of the solid test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 µI of 0.9% w/v sodium chloride solution was added for wetting of the test item. Duplicate tissues, treated with 50 µI of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 IJI of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.

At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca2+ and Mg2+. 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 item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.

2.2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours ±5 minutes at room temperature in a biological safety cabinet ensuring that the plates were protected from light. 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 taken using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 ml micro tubes containing 850 IJI of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-Ioaded tissues.

Absorbance/Optical Density Measurements (Day 2):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.

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 tJl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 tJl 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 vivo

Results

Irritation parameter:

overall irritation score

Remarks on result:

other: Not applicable as an in vitro method was used. The test item was considered to be Non-Corrosive to the skin.

Irritant / corrosive response data:

Direct MTT Reduction: The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

Any other information on results incl. tables

The relative mean viability of the test item treated tissues was as follows:

240 minutes exposure = 98%

60 minutes exposure = 106%

3 minutes exposure = 106.5%

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was 3.5% relative to the negative control treated tissues following the 240-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.200. The negative control acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Conclusion: The test item was considered to be Non-Corrosive to the skin. The test item was also classified as non-corrosive to the skin according to EU labelling regulations Commission Directive 2001/59/EC. No label is required. The UN packing group Non-Corrosive is required.

Executive summary:

Introduction:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to be compatible with the following:

-       OECD Guideline for the Testing of Chemicals No. 431 "In Vitro Skin Corrosion: Human Skin Model Test" (adopted 13 April 2004)

-        Method B.40 of Commission Regulation (EC) No. 440/2008 The EPISKIN™ model 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 I) and R34 (UN packing group II & III) test items.

Methods:

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 24:0 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-Ioading. 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 IJI samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 540 nm (OD540).

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:

240 minutes exposure : 98%

60 minutes exposure : 106.0%

3 minutes exposure : 106.5%

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

Conclusion: The test item was considered to be Non-Corrosive to the skin. The test item was also classified as non-corrosive to the skin according to EU labelling regulations Commission Directive 2001/59/EC. No label is required. The UN packing group Non-Corrosive is required.