[1,1'-Biphenyl]-2,2'-diol, 3,3'-bis(1,1-dimethylethyl)-5,5'-dimethoxy-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was performed between 03 March 2009 and 05 March 2009.

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 the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: SkinEthic Reconstituted Human Corneal model

Deviations:

no

Principles of method if other than guideline:

The SkinEthic RHC model consists of transformed human keratinocytes 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. Test materials are applied directly to the culture surface, at air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.
The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.
Cytotoxicity was determined by the reduction of MTT (3 [4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test material treated tissues (quantitative measurement of tissue viability) relative to the negative control.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/08 Date of Signature: 04/03/09

Test material

Test material information

Constituent 1

Reference substance name:

L17_Precursor

IUPAC Name:

L17_Precursor

Details on test material:

Sponsor's identification : L17_Precursor
Description : pale yellow powder
Batch number : DALA046312
Date received : 11 November 2008
Storage conditions : room temperature in the dark

Test animals / tissue source

Species:

other: SkinEthic Reconstituted Human Corneal model

Strain:

other: SkinEthic Reconstituted Human Corneal model

Details on test animals or tissues and environmental conditions:

Not applicable, no animals used within this study.

Information on SkinEthic Reconstituted Human Corneal model:
Supplier : SkinEthic Laboratories, Nice, France
Date Received : 03 March 2009

On arrival, the SkinEthic HCE tissues (Day 6), 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 material and negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the Day 7 tissues were transferred from the ‘arrival plates’ into the wells of the ‘treatment plates’ containing the maintenance medium.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Triplicate tissues were treated with 30 µl of solution A to serve as negative controls and triplicate tissues were treated with 30 µl of 1 % w/v SDS to serve as positive controls.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
30 mg of the test material


VEHICLE
Not applicable

Duration of treatment / exposure:

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.

Observation period (in vivo):

The MTT loading plate was placed into an incubator for approximately three hours.

After 1st visual observation, allowed to stand overnight ready for the optical density observation.

Number of animals or in vitro replicates:

Not applicable

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
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 material. 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 approximately three hours at 37°C, 5% CO2 in air.

- Time after start of exposure:
10 minutes

SCORING SYSTEM:
The mean OD540 values of the duplicate tissues were calculated. Each of these OD540 values had already been corrected for blanks by the microplate reader.
The relative mean tissue viabilities (% of the negative control) were calculated as follows:

Relative mean tissue viability = (mean OD540 of test material / mean OD540 of negative control) x 100

The mean tissue viabilities for the test material was compared to the respective untreated negative control and classified according to the following table:

Relative Mean tissue viability
(% negative control) Prediction
Tissue viability <60 Irritant (I)
Tissue viability ≥60 Non-Irritant (NI)


TOOL USED TO ASSESS SCORE:
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 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.

Results and discussion

In vitro

Results

Remarks on result:

other:

Remarks:

See table in "Any other information on results.."

Any other information on results incl. tables

Table 1              Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material	Mean Tissue Viability	Mean OD540	% Viability
Negative Controlφ	0.950	0.976	100*
	1.001
Positive Controlφ	0.770	0.731	74.9
	0.692
Test Material	1.057	1.062	108.8
	1.066

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

φ =    Control group shared with Project number 1571/0071

 

Table 2              Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Score
	Tissue 1	Tissue 2
Negative ControlÅ	-	-
Positive ControlÅ	+	+
Test Material	-	-

MTT Visual Scoring Scheme of SkinEthic Tissues

-     =  Blue tissue (viable)

+    =  Blue/White tissue (semi viable)

++  =  Tissue completely white (dead)

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

According to the protocol followed the test material 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 material using the SkinEthic Reconstituted 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 by the test material followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test material 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 1% 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 (OD₅₄₀). Data are presented in the form of % viability (MTT conversion relative to negative controls).

The test material was classified according to the following criteria:

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

ii)                 If the % relative mean tissue viability was < 60% the test material was considered to be an irritant.

Results. 

The relative mean viability of the test material treated tissues after a 10 minute exposure was 108.8%.

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 protocol followed the test material was considered to be a Non‑Irritant (NI).