Reaction product of {Sulfonic acid derivatives of [4-(aminoalkylamino)-6-substituted-1,3,5-triazin-2-ylamino]benzene}, ammonia water, sodium chloride and [poly(1~4)chlorosulfonyl(poly(1~4) benzo-poly(3~0)pyrido-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:

Experimental starting date: 10 September 2013; Experimental completion date 16 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to the Assessment of Skin Irritation Potential using the EPISKIN Reconstructed Human Epidermis Model and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: CIM-25
Batch: MB-1
Expiry: 31 March 2014
Storage Conditions: ambient 10 to 30 °C

Test animals

Species:

other: EPISKIN ™ in vitro Reconstructed Human Epidermis (RHE) Model

Strain:

other: No applicable

Details on test animals or test system and environmental conditions:

Not applicable for test animals.

EPISKIN ™ Reconstructed Human Epidermis Model Kit supplied by SkinEthic Laboratories, Lyon, France

Test system

Type of coverage:

other: Not applicable

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Amount / concentration applied:

10 mg of the test item was applied to epidermal surface.

Duration of treatment / exposure:

15 minute exposure period

Observation period:

42 hours post exposure incubation period.

Number of animals:

Not applicable

Details on study design:

TEST ITEM FORMULATION AND EXPERIMENTAL PREPARATION
The test item was used as supplied.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS, MTT AND ACIDIFIED ISOPROPANOL
The negative control item, PBS, was used as supplied.
The positive control item, SDS, was prepared as a 5% w/v aqueous solution.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Negative Control:
Identification: Phosphate Buffered Saline Dulbecco's (PBS) with Ca++ and Mg++

Positive Control:
Identification: Sodium Dodecyl Sulphate (SDS)

PRE-TEST PROCEDURE
ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT:
MTT Salt Metabolism, Cell Viability Assay:

The MTT assay, a colorimetric 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 blue formazan salt 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 is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:

10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues. This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test substance like viable tissues.

Water-killed tissues were prepared by placing untreated EPISKIN™ tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37°C, 5% CO2 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30°C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, the MTT reducing test item was applied to three water-killed tissues. In addition, three water-killed tissues remained untreated. The untreated water-killed controls showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:

Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes

2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37°C, 5% CO2 in air overnight.

MAIN TEST
APPLICATION OF TEST ITEM AND RINSING (DAY 1)
2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12-well plate.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL sterile distilled water was topically applied to the epidermal surface in order to improve further contact between the test item and the epidermis. 10 mg of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.

Due to the intrinsic 'blue' color of the test item a further limitation of the assay is possible color interference with measurement of the extracted MTT. Therefore in addition to the application procedure described above, three additional test item treated tissues and two additional color correction negative control tissues (DPBS) were treated to evaluated possible interference. These tissues followed the same treatment steps as the viable tissues except for the MTT step where MTT incubation is replaced by incubation with fresh assay medium for the test item treated color correction tissues. The additional negative control tissues were incubated in MTT.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.

MTT LOADING/FORMAZAN EXTRACTION (DAY 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30°C for possible inflammatory mediator determination.

2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. 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 made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of
formazan crystals out of the MTT-loaded tissues.

Since it was possible that the colored test item may have caused color interference, three test item treated tissues were used for MTT color correction purposes. The tissues were transferred to 12-well plates containing 2 ml of assay medium in a sufficient number of wells. Similarly two color correction negative control tissues treated with DPBS were also transferred to 12-well plates containing 2 ml of assay medium in a sufficient number of wells for determining and correcting background color in the tissues. These tissues were then treated identically to the tissues placed in the MTT solution.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.

For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 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 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Relative Mean Viability %

Value:

99.2

Remarks on result:

other:

Remarks:

Basis: mean (test item). Time point: 15-minute exposure period and 42 hours post-exposure incubation. Remarks: Non-Irritant. (migrated information)

In vivo

Irritant / corrosive response data:

Test Item, Positive Control Item and Negative Control Item
The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1.. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test item treated tissues was 99.2% after a 15-Minute exposure period and 42 hours post-exposure incubation period.

It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Quality Criteria:
The relative mean tissue viability for the positive control treated tissues was 7.2% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.9%. The positive control acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.829 and the standard deviation value of the percentage viability was 3.9%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 4.3%. The test item acceptance criterion was therefore satisfied.

Other effects:

Direct MTT Reduction:
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed no degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Color Interference Check:
Before dosing was due to commence it was decided that color correction tissues should be incorporated into the testing due to the intrinsic 'dark blue' color of the test item. The color correction tissues would be used to measure any staining, adherence or absorbed test item into the tissue that may have caused interference with the optical density measurements.

Therefore in addition to the normal dosing procedure, three tissues were treated with the test item. A further two tissues remained untreated for correcting background color. These additional groups were not placed into MTT but into assay medium for the equivalent MTT exposure of 3 hours.

The optical density results indicated that negligible color interference had occurred due to the colored test item and therefore the additional tissues were not required for correction of the results.

Any other information on results incl. tables

Table 1: Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item	OD562 of Tissues	Mean OD562 of Triplicate Tissues	± SD of OD562	Relative Individual Tissue Viability (%)	Relative Mean Viability (%)	± SD of Relative Mean Viability (%)
Negative Control Item	0.843	0.829	0.032	101.7	100*	3.9
	0.852			102.8
	0.792			95.5
Positive Control Item	0.051	0.059	0.016	6.2	7.2	1.9
	0.049			5.9
	0.078			9.4
Test Item	0.793	0.822	0.036	95.7	99.2	4.3
	0.862			104.0
	0.812			97.9

 

 

SD = Standard deviation

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

 

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 classified as non-irritant. The following classification criteria apply:

ED DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 cannot be determined).

Executive summary:

Introduction

 

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis 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 reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-Iα in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.

 

 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 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-labeled 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-labeled 96-well plate. The optical density was measured at 562 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 99.2% after the 15-Minute exposure period and 42 hours post-exposure incubation period.

 

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

 

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

ED DSD & CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 cannot be determined).