Reaction product of 3-[4-(2-aminoethylamino)-6-(4-sulfoanilino)-1,3,5-heterocycle-2-ylamino]benzensulfonic acid, ammonia water, sodium chloride and {mixture of [poly(1~4)chlorosulfonylphthalocyaninato-N29,N30,N31,N32]copper(II), [poly(1~3)chlorosulfonyl (tribenzo[b,g,l]pyrido[2,3-q]-5,10,15,20-tetraazaporphyrinato-N21,N22,N23,N24)]copper(II),[poly(1~2)chlorosulfonyl(dibenzol[b,g(or b,l)dipyrido[2,3(or 3,2)-l,q (or g,q)]-5,10,15,20-tetraazaporphyrinato-N21,N22,N23,N24)] copper(II) and [monochlorosulfonyl(benzo[b]tripyridol[2,3(or 3,2)-g,lq]-5,10,15,20- tetraazaporphyrinato- N21,N22,N23,N24)] copper(II)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 March 2009 to 05 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 (HCE, SkinEthic Laboratories, Nice, France)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19 August 2008; Date of signature: 04 March 2009

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification : H-CB sodium salt
Description : blue solid
Lot number : MB-2
Date received : 04 December 2008
Storage conditions : room temperature in the dark

Test animals / tissue source

Species:

other: Reconstituted human corneal epithelial tissue.

Test system

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

10 minute exposure and 3 hour incubation period.

Results and discussion

In vitro

Results

Irritation parameter:

other: Percentage tissue viability

Value:

104

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Assessment of Direct Test Material Reduction of MTT

The test material was not able to directly reduce MTT.

Due to the blue colour of the test material it was not possible to determine if the test material directly reduced MTT. Therefore, as a precaution, killed control tissues were included in the assay to quantitatively correct of the results of the MTT assay if necessary. However the killed tissues demonstrated no absorbancy and therefore the results of the killed tissues were not used for reporting purposes.

Assessment of eye Irritation Potential

The mean OD₅₄₀ values and mean viabilities for each treatment group are given in Table 1 (please see attached background material).

The relative mean viability of the test material treated tissues after a 10 minute exposure was 104.1%

It was considered unnecessary to proceed with tissue histopathology.

Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment)

The qualitative evaluation of tissue viability is presented in Table 2 (please see attached background material).

The test material and negative control treated tissues appeared blue which was considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: 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.

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, Nice, France) 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 were 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 104.1%.

It was considered unnessary 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).