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)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 February 2009 to 09 February 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 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: Determination of skin irritation potential using the EPISKIN (TM) Reconstituted Human Epidermis Model

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

Species:

other: Reconstituted human epidermis

Test system

Type of coverage:

other: Topical

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

15 minutes exposure and 42 hours post exposure incubation.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

102.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Direct MTT Reduction

Due to the blue colour of the test material it was not possible to determine if the test material directly reduced the MTT. Thereforem 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.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are gven in Table 1 (attached background material). The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 102.6% after a 15 minute exposure.

The qualitative evaluation of tissue viability is gven in Table 2 (attached backgound material).

Following the 15 minute exposure the test material treated tissues appeared blue which was considered indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was < 40% relative to the negative control treated tissues and the Standard Deviation (SD) value of the % viability was < 20%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative contol treated tissues was > 0.6 and the SD value of the % viability was < 20%. 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: not specified

Conclusions:

The test material was considered to be Non-Irritant.

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TM reconstituted human epodermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourmetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5 -dimethylthiazol-2 -yl]-2,5 -diphenyl-tetrazolium bromide) to a blue formazon salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL - 1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-orrotant result.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. 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 μl 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 % viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: The relative mean viability of the test material treated tissues was 102.6% after a 15 minute exposure.

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

Conclusion: The test material was considered to be non-irritant.