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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 18 January 2011 and 20 January 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the 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: The guideline used is adapted from the references listed in the overall remarks section.

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: pellicier.

- Physical state: White solid.

- Analytical purity: 94%.

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

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

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

Test animals / tissue source

Species:

other: SkinEthic HCE model consists of transformed human corneal epithelial cells

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

The SkinEthic HCE model consists of transformed human corneal epithelial cells of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum free and chemically defined medium.

Test system

Vehicle:

other: undiluted

Controls:

other: (negative control) Solution A, Composition for 1 Litre: Na2HPO4 0.142 g/l, Glucose 1.802 g/l, HEPES 7.149 g/l, KCl 0.224 g/l and NaCl 7.597 g/l. (positive control) The positive control item, Sodium Dodecyl Sulphate (SDS) 2% w/v, was prepared in sterile di

Amount / concentration applied:

The test item is applied directly to the culture surface, at the air interface (undiluted and/or end use dilutions was tested directly).

Duration of treatment / exposure:

10 minutes.

Observation period (in vivo):

(Incubation period of 3 hours)

Number of animals or in vitro replicates:

(Triplicate tissues)

Details on study design:

Pre-Test
Assessment of Direct Test Item Reduction of MTT: One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test item is only a problem, if at the time of the MTT test (after the chemical has been rinsed off) there are still sufficient amounts of the test item on or in the tissues. To identify this possible interference, the test item was checked for its ability to reduce MTT directly.
30 mg of test item was added to 1 ml of a 0.5 mg/ml MTT solution and incubated at room temperature in the dark for 3 hours. Untreated MTT solution was used as a control. If the MTT solution turned blue, the test item was presumed to have reduced the MTT.

Receipt of Tissues: On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 300 µl of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37°C, 5% CO2 in air.

Preparation of Tissues: Using sterile techniques, 1 ml of maintenance medium at room temperature, was dispensed into the appropriate number of wells of 6-well plates designated ‘treatment plates’. Each well was labelled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test item, negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the 7 Day old tissues were transferred from the ‘arrival plates’ into the wells of the ‘treatment plates’ containing the maintenance medium.

Main Test: Triplicate tissues were treated with 30 mg of the test item for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 µl of solution A to serve as negative controls and triplicate tissues were treated with 30 µl of 2% w/v SDS to serve as positive controls. The plates were incubated at 37°C, 5% CO2 in air during the exposure time.
At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS). Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24 well plate designated ‘holding plate’ containing 300 µl of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per group) were transferred to a pre labelled 24 well plate designated ‘MTT Loading plate’ containing 300 µl of a 0.5 mg/ml MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for three hours at 37°C, 5% CO2 in air.
At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were rinsed twice with phosphate buffered saline and blotted on absorbent paper to remove residual MTT solution and transferred to a pre labelled 24 well plate designated ‘MTT extraction plate’ containing 0.75 ml of Isopropanol in each of a sufficient number of wells. An extra 0.75 ml of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.
At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µl tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl of Isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.

Tissue Histology: One tissue for each treatment group was retained for possible tissue histopathology.
The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a pre-labelled 1.5 ml Eppendorf tube containing 1 ml of 10% Formalin and stored at room temperature.

Results and discussion

In vivo

Results

Irritation parameter:

other: tissue viability

Basis:

other: tissue viability

Time point:

other: 10 minutes

Score:

108.1

Max. score:

1.046

Reversibility:

other: Not applicable

Remarks on result:

other: MTT Assay for tissue cell vibility

Irritant / corrosive response data:

Assessment of Direct Test Item Reduction of MTT: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Assessment of Eye Irritation Potential: The individual and mean OD540 values and mean viabilities for each treatment group are given in Table in 'Any other information on results incl. tables'.
The relative mean viability of the test item treated tissues after a 10 Minute exposure period was 108.1%.
It was considered unnecessary to proceed with tissue histopathology.

Assay Acceptance Criterion: The quality criterion required for the acceptance of results in the test was satisfied.

Any other information on results incl. tables

Table Assessment of Eye Irritation Potential – Viability of HCE Tissues

Item	OD540of Individual Tissue	Mean OD540	Relative Mean Viability (%)
Negative Control	0.934	0.951	100*
	0.968
Positive Control	0.449	0.449	47.2
	0.449
Test Item	1.009	1.028	108.1
	1.046

 

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

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: criteria of Van Goethem et al., 2006, Nguyen DH et al., 2003.

Conclusions:

According to the study plan followed the test item was considered to be a Non-Irritant (NI).

Executive summary:

Introduction. The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic Reconstructed Human Corneal model (HCE, SkinEthic Laboratories,,) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods. The experimental design of the study consists of a test for direct reduction of MTT (3‑[4,5‑dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test item for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 2% w/v Sodium Dodecyl Sulphate served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD₅₄₀). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test item was classified according to the following criteria:

i)  If the percentage relative mean tissue viability was ≥60% the test item was considered to be non‑irritant (NI).

ii) If the percentage relative mean tissue viability was <60% the test item was considered to be an irritant (I).

Results. The relative mean viability of the test item treated tissues after a 10‑Minute exposure period was108.1%.

It was considered unnecessary to proceed with tissue histopathology.

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

Conclusion. According to the study plan followed the test item was considered to be a Non‑Irritant (NI).