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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 March 2011 to 01 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study conducted to EU & 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:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), ANNEX III, B.46. IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST
Qualifier:
according to
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F03-0318

Test animals

Species:
human
Strain:
not specified
Details on test animals and environmental conditions:
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-013
Expiry date: 04 April 2011

Test system

Type of coverage:
open
Preparation of test site:
other: Not applicable
Vehicle:
water
Controls:
yes
Amount / concentration applied:
First, 10 µl distilled water (Supplier: HungaroPharma; Batch No.: 803 0211; Exp. date: 22 August 2011) was applied to the epidermal surface (in order to improve further contact between powder and epidermis) and then 10 mg of the test item was applied evenly to the epidermal surface.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes
Observation period:
42 hours
Number of animals:
Not applicable
Details on study design:
Preparation of the Test Item
The test item was applied in its original form, no formulation was required.

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
SDS 5 % aq. solution (prepared in LAB Research Ltd.):
Sodium Dodecyl Sulphate (SDS)
Supplier: SIGMA-ALDRICH
Batch No.: 129K0187
Expiry date: December 2012 Distilled water
Supplier: Hungaropharma Zrt
Batch No.: 803 0211
Expiry date: 22 August 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).

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 Content
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-MAIN3-013; Exp. Date: 06 April 2011)
A flask of sterile “Assay Medium” for use in for use in MTT assays.(Batch No.: 11-ESSC-013; Exp. Date: 06 April 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. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed below.

Check-method for possible direct MTT reduction with test substance
An amount of 10 mg 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, three additional chemical-treated tissues were used for the non specific OD evaluation. These tissues 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 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)
Test Item
First, 10 µl distilled water (Supplier: HungaroPharma; Batch No.: 803 0211; Exp. date: 22 August 2011) was applied to the epidermal surface (in order to improve further contact between powder and epidermis) and then 10 mg of the test item was applied evenly to the epidermal surface. Test substance was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Positive and negative control
An amount of 10 µl positive (SDS) or negative (PBS) control were added to each of three test skin units by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Additional controls for staining effects
These tissues followed the same application procedure as the other Test Item treated tissues.
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 3 staining controls were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The three 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 vivo

Irritant / corrosive response data:
VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.639. The positive control result showed 9 % 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, three additional chemical-treated tissues were used for the non specific OD evaluation. Mean OD (measured at 540 nm) of these tissues were determined as 0.018, Non Specific Colour % was calculated as 2.76%. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Any other information on results incl. tables

Cell VIABILITY

 

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

0.668

104

2

0.642

100

3

0.608

95

mean

0.639

100

standard deviation (SD)

4.71

Positive Control:

SDS 5% aq. solution

 

1

0.080

13

2

0.041

6

3

0.051

8

mean

0.058

9

standard deviation (SD)

3.18

Test Item:

Reactive Red F03-0318

 

1

0.565

88

2

0.534

83

3

0.484

76

mean

0.528

83

standard deviation (SD)

6.39

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 F03-0318 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), COMMISSION REGULATION (EC) No 761/2009 ANNEX III, B.46 and the Principles of Good Laboratory Practice (GLP) and reported with a valid GLP certificate.

 

Disks of EPISKIN (three units / chemical) were treated with Reactive Red F03-0318 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 atin 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 F03-0318 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.