L-Lysine, N2,N6-bis[N-(1-oxododecyl)glutamyl]-, sodium salt (1:?)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 December 2010 to 10 December 2010.

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Date of inspection: 20 July 2010 Date of Signature: 29 October 2010)

Test material

Test material information

Constituent 1

Reference substance name:

Pellicer

IUPAC Name:

Pellicer

Details on test material:

- Name of test material (as cited in study report): pellicer.

- Physical state: white solid.

- Lot/batch No.: 01-09-17.

- Analytical purity: not supplied.

- Expiration date of the lot/batch: 31 December 2011.

- Storage condition of test material: Room temperature in the dark over silica gel.

Test animals

Species:

other: EPISKINTM in vitro Reconstituted Human Epidermis (RHE) Model

Strain:

other: EPISKINTM in vitro Reconstituted Human Epidermis (RHE) Model

Details on test animals or test system and environmental conditions:

(In vitro test system was used.)


TEST ANIMALS
- Source: SkinEthic Laboratories, Nice, France.
The EPISKINTM model is a three-dimensional reconstituted 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.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Warmed to approximately 37ºC (assay medium).

Test system

Type of coverage:

other: Topical

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable, no control was set for the in vitro test system used in this study.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg of the solid item, undiluted.
- 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.


VEHICLE
Not applicable.

Duration of treatment / exposure:

3, 60 and 240 minutes.

Observation period:

Not applicable.

Number of animals:

(Duplicate tissues)

Details on study design:

Pre-Test

Assessment of Direct Test Item Reduction of MTT

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-diphenyl-tetrazolium bromide) to a purple 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 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/purple relative to the control, the test item was presumed to have reduced the MTT.


Pre-Incubation (Day 0: tissue arrival)
2.2 ml of maintenance medium, warmed to approximately 37ºC, 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 37ºC, 5% CO2 in air for at least 24 hours. After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.


Main Test

Application of Test Item and Rinsing (Day 2)
2.2 ml of assay medium, warmed to approximately 37ºC, 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 µl of 0.9% w/v sodium chloride solution was added for wetting of the test item. Duplicate tissues, treated with 50 µl of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µl 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 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.

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 EPISKINTM 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 µl 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 loaded tissues.

Absorbance/Optical Density Measurements (Day 3)
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 µl samples were transferred to the appropriate wells of a pre-labelled 96 well plate. 200 µl 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

Results 1

Irritation / corrosion parameter:

other: other: Viability of cells

Value:

85.7

Remarks on result:

other:

Remarks:

Basis: mean Viability of cells. Time point: 240 minutes. Remarks: unit of score - %. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Viability of cells

Value:

84.8

Remarks on result:

other:

Remarks:

Basis: mean Viability of cells. Time point: 60 minutes. Remarks: unit of score - %. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: Viability of cells

Value:

82.7

Remarks on result:

other:

Remarks:

Basis: mean Viability of cells. Time point: 3 minutes. Remarks: unit of score - %. (migrated information)

In vivo

Irritant / corrosive response data:

The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

Other effects:

The relative mean tissue viability for the positive control treated tissues was 6.9% 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 0.231. 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:

The test item was considered to be Non-Corrosive to the skin.

Executive summary:

Introduction:

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

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

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) chemicals.

Methods:

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 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 ~I 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: PAGE 6

240 minutes exposure 85.7%

60 minutes exposure 84.8%

3 minutes exposure 82.7%

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.