Alkali salt of substituted aryl sulfonyl triazinyl amino hydroxy sulfonyl aryl diazo naphthalene sulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 January 2011 to 07 January 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD test guidance in compliance with GLP and reported with a valid GLP certificate.

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: –OECD Guidelines for Testing of Chemicals, Section 4, No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” adopted 22 July 2010.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Name: Reactive Red F08-0146

Test animals

Species:

other: EPISKIN reconstituted human epidermis

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM

Human Skin
EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1984). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Source: Skinethic, Nice, France.
Batch No.: 11-EKIN-001
Expiry date: 10 January 2011

Quality Control
EPISKIN-SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

Test system

Type of coverage:

open

Preparation of test site:

other: Not applicable

Vehicle:

water

Controls:

yes

Amount / concentration applied:

10 mg of test item was applied evenly to the epidermal surface of each of the three test skin units, then 30 µl distilled water was added to the test item to ensure good contact with the epidermis.
10 µl PBS was added to each of the three negative control skin units
10 µl SDS was added to each of the three positive control skin units
For additional controls for staining effects of the test item, 10 mg of test item was applied evenly to the epidermal surface of both skin units, then 30 µl distilled water was added to the test item to ensure good contact with the epidermis.

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (18-28°C).

Observation period:

After the 42 hours incubation all EPISKIN-SM units except the 2 staining controls were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The two additional controls for coloured substances were transferred to wells filled with fresh assay medium. Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

Number of animals:

Not applicable

Details on study design:

The irritation potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD 404).
The test is designed to predict and classify the skin irritant potential of chemicals according to chemical safety regulations, using the reconstructed human epidermis model EpiSkinTM small model and parameters related to skin irritation.

EpiSkinTM Small Model (EpiSkinTMSM) is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller C. et al., 2002, Mosmann T., 1983). The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. The % reduction in viability is used to predict the irritation potential.

Controls
Positive and negative controls, furthermore additional controls for coloured substance were included in the experiment.

Negative Control
Phosphate Buffered Saline (PBS):
Supplier: SIGMA-ALDRICH
Batch No.: BCBC6808
Expiry date: December 2014

Positive Control
Sodium Dodecyl Sulphate (SDS) 5% aq. solution (prepared by LAB Research Ltd.):
Sodium Dodecyl Sulphate (SDS)
Supplier: SIGMA-ALDRICH
Batch No.: 129K0187
Expiry date: December 2012
Distilled water
Supplier: TEVA Magyarország Zrt
Batch No.: 7530810
Expiry date: August 2013

Justification for Selection of the Test System
The EPISKIN model has been validated for irritation testing in an international trial, it is considered to be suitable for this study.

Demonstration of Proficiency
Prior to routine use of the method LAB Research Ltd. demonstrated technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

Kit Contents
Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” for incubations. (Batch No.: 11-MAIN3-001; Exp. Date: 12 January 2011)
A flask of sterile “Assay Medium” for use in for use in MTT assays.(Batch No.: 11-ESSC-001; Exp. Date: 12 January 2011)

Number of Replicate Wells
In this assay 3 replicates for the test item and 3 negative controls + 3 positive controls were used. Furthermore 2 replicates of additional controls for coloured substance were used.

Kit Reception Quality Check
The colour of the agar medium used for transport was checked for its pH:
-orange colour = good
-yellow or violet colour = not acceptable

The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C:
-the indicator changes from white to grey at 40°C

The kit was found to be in good order at reception.

Storage
The EPISKIN-SM kit was kept in its packaging at room temperature and the assay and maintenance medium supplied with the kit was stored at 2-8°C until the initiation of the test.

Additional materials

MTT stock solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was dissolved to a final concentration of 3 mg/mL in saline buffer (PBS). The obtained stock solution can be stored in a refrigerator (2-8°C), protected from light up to 15 days.

MTT ready to use solution
The MTT stock solution was diluted with pre-warmed “assay medium” to a final concentration of 0.3 mg/mL. The obtained solution was used within one hour.

Acidified isopropanol
Isopropanol was diluted with HCl acid to a final concentration of 0.04N HCl.
The obtained solution can be stored in a refrigerator (2-8°C), protected from light for one month.

Indicator for potential false viability
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test substance is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls will be used to detect and correct for test substance interference with the viability measurement.

Check-method for possible direct MTT reduction with test substance
Approximately 10 µL or 10 mg of test item was added to 2 mL MTT ready to use solution and mixed. The mixture was incubated for three hours at room temperature protected from light and then any colour change was assessed:
- Test substances which do not interact with MTT: yellow
- Test substances interacting with MTT: blue or purple

If the MTT solution colour becomes blue or purple, the test substance interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non specific reduction of the MTT (i.e. by using killed epidermis).

The test item showed no direct interaction with MTT.

Check-method to detect the colouring potential of test-substances
Prior to treatment, chemicals were evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
As the test item has an intrinsic colour, further evaluation to detect colouring potential was not necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test substance to stain the epidermis by using additional control tissues.

Additional control(s) for dyes and chemicals able to colour the tissue
In addition to the normal procedure, two additional chemical-treated tissues were used for the non specific OD evaluation. This tissue followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD readings were made following the same conditions as for the other tissues.

Performance of the study
Procedures described in sections below were performed under axenic conditions.

Pre-incubation (day [-1])
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2.

Application and rinsing (day 0)
10 mg of test item was applied evenly to the epidermal surface of each of the three test skin units, then 30 µl distilled water* was added to the test item to ensure good contact with the epidermis.
10 µl PBS was added to each of the three negative control skin units
10 µl SDS was added to each of the three positive control skin units
For additional controls for staining effects of the test item, 10 mg of test item was applied evenly to the epidermal surface of both skin units, then 30 µl distilled water* was added to the test item to ensure good contact with the epidermis The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (18-28°C).

After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1x solution (0.9 %) to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source care was taken to avoid the damage of epidermis).

After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2.

MTT test after 42 hours incubation (day 2)
After the 42 hours incubation all EPISKIN-SM units except the 2 staining controls were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The two additional controls for coloured substances were transferred to wells filled with fresh assay medium. Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

Formazan extraction (day 2)
At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (day 2)
Following the formazan extraction, 2×200 µL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer was read at 540 nm using acidified isopropanol solution blank (6×200 µL).

Note: The validity of the microplate reader was verified with a standard verification plate daily before use. The standard plate was calibrated yearly by the manufacturer

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: optical density

Value:

0.746

Remarks on result:

other:

Remarks:

Basis: mean. Time point: overall. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:

CALCULATIONS OF VIABILITY PERCENTAGES

Data calculation for normal test substances
Blank:
The mean of the 6 blank OD values was calculated
Negative control:
Individual negative control OD values were corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
The corrected mean OD of the 3 negative control values were calculated: this corresponds to 100% viability
Positive control:
Individual positive control OD values were corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
The corrected mean OD of the 3 positive control values were calculated
The % viability for each positive control replicate was calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
The mean value of the 3 individual viability % for positive control was calculated:
Mean PC % = (%PC1 +%PC2 +%PC3) / 3
Test substance:
Individual test substance OD values were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
The corrected mean OD of the 3 test substance values were calculated
The % viability for each test substance replicate was calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
The mean value of the 3 individual viability % for test substance was calculated
Mean TT % = (%TT1 +%TT2 +%TT3) / 3
Data calculation for MTT-interacting substances

Test substances that interfere with MTT can produce non specific reduction of the MTT. It is necessary to evaluate the OD due to non specific reduction and to subtract it before calculations of viability %.

Non specific MTT reduction calculation (NSMTT):
NSMTT = [(ODKT- ODKU) / ODNC] × 100
ODKU: untreated killed tissues OD
ODKT: test substance treated killed tissues OD
ODNC: negative control OD

If NSMTT is > 30% relative to the negative control: additional steps must be undertaken if possible, or the test substance must be considered as incompatible with the test.

True MTT metabolic conversion (TODTT) is undertaken if NSMTT is < 30%:
TODTT = [ODTT – (ODKT – ODKU)]
ODTT: test substance treated viable tissues

The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2= [TODTT1 / mean ODNC] × 100
% RV 3 = [TODTT1 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test substance is calculated
Mean Relative Viability % = (% RV 1 +% RV 2 +% RV 3) / 3

Data calculation for dyes and chemicals able to colour the tissue
For test substances detected as able to stain the tissues the non specific OD is evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability %.

Non Specific Colour % (NSC%):
NSC % = (ODCT / mean ODNC) × 100
ODCT: test substance treated tissue (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

If NSC % is ≤ 5% then the normal calculation mode is used (see 3.7.1).
If NSC % is > 30% relative to the negative control, additional steps must be undertaken if possible, or the test substance must be considered as incompatible with the test.
v True MTT metabolic conversion (TODTT) is undertaken if NSC % is > 5% and ≤ 30%
TODTT = [ODTV - ODCT]
ODTV: test substance treated tissue (incubated with MTT)
ODNC: negative control OD (incubated with MTT)

The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2= [TODTT1 / mean ODNC] × 100
% RV 3 = [TODTT1 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test substance is calculated
Mean Relative Viability % = (% RV 1 +% RV 2 +% RV 3) / 3

Other effects:

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.795. The positive control result showed 25 % viability. Each standard deviation value (SD) of the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY

Possible direct MTT reduction with test substance
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test substances
As the test item has an intrinsic colour, two additional chemical-treated tissues were used for the non specific OD evaluation. Mean OD (measured at 540 nm) of these tissues were determined as 0.024, Non Specific Colour % was calculated as 2.96 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Disks of EPISKIN (three units / chemical) were treated with Reactive Red F08-0146 and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution (0.9%). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.
SDS 5% and PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a % relative to negative control.
The test substance is considered to be irritant to skin, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

Any other information on results incl. tables

The results of the optical density (OD) measured at 540 nm of each replicate and the calculated % viability of the cells is presented below:

 

Substance	Optical Density (OD)		Viability (%)
Negative Control: PBS	1	0.859	108
	2	0.661	83
	3	0.865	109
	mean	0.795	100
	standard deviation (SD)		14.59
Positive Control: SDS	1	0.176	22
	2	0.197	25
	3	0.223	28
	mean	0.198	25
	standard deviation (SD)		11.86
Test Item: Reactive Red F08-0146	1	0.732	92
	2	0.720	91
	3	0.785	99
	mean	0.746	94
	standard deviation (SD)		4.64

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this in vitro skin irritation test in the EPISKIN model with Reactive Red F08-0146 the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

Executive summary:

This study has been performed in accordance with the study plan the OECD Guidelines for Testing of Chemicals (No. 439, 22 July 2010), EpiSkin™ SOP, Version 1.8 (February 2009) and the Principles of Good Laboratory Practice (GLP) and reported with a valid GLP certificate.

 

Justification for Selection of the Test System

The EPISKIN model has been validated for irritation testing in an international trial, it is considered to be suitable for this study.

 

Disks of EPISKIN (three units / chemical) were treated with Reactive Red F08-0146 and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution (0.9%). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 degree C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.

SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a % relative to negative control.

The test substance is considered to be irritant to skin, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

 

In this in vitro skin irritation test in the EPISKIN model with Reactive Red F08-0146 the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

 

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

 

Demonstration of proficiency

Prior to routine use of the method LAB Research Ltd. demonstrated technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.