[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February from the 12th to the 14th, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

Version / remarks:

26 July 2013.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

Version 1.8 (February 2009)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

The test item was applied in its original form, no formulation was required.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France.
- Tissue batch number: 14-EKIN-004
- Cell source: 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., 1994).
- Manufacture: EpiSkinTMSM kits are manufactured according to defined quality assurance procedures (certified ISO 9001).
- Safety: all biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.
- Storage: the EpiSkinTMSM units were kept in their packaging at room temperature until the preincubation was started. The maintenance and assay medium were stored at 2-8 °C.

REMOVAL OF TEST MATERIAL: after the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

CONTROLS
- Replicates: three replicates were used.
- Application: 10 μl positive control or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT stock solution: MTT was dissolved to a final concentration of 3 mg/ml in saline buffer (PBS).
- Storage of stock solution: the obtained stock solution can be stored in refrigerator (2-8 °C), protected from light up to 15 days.
- MTT concentration: 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.
- Incubation time: an amount of 10 mg test item was added to 2 ml MTT 0.3 mg/ml solution and mixed. The mixture was incubated for three hours at 37 °C protected from light and then any colour change observed.
- Interation evaluation: if the MTT solution colour becomes blue or purple, the test substance interacts with the MTT. It is then necessary to evaluate the part optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed epidermis).
- MTT test after 42 hours incubation: after the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells and then incubated for 3 hours (± 5 min) at 37 °C in an incubator with 5 % CO2 protected from light.

ADDITIONAL CONTROL
- Non specific OD evaluation: one additional chemical-treated tissue was used for the non-specific OD evaluation.
- Ttreatment: the 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.
- Reading: OD reading was made following the same conditions as for the other tissues.

FORMAZAN EXTRACTION
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from the unit using a biopsy punch. 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. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μl sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at 570 nm using acidified isopropanol solution as the blank (6×200 μl).

ASSAY ACCEPTANCE CRITERIA
The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-40 % and the standard deviation value (SD) of the % viability should be ≤ 18.
For test chemicals, the standard deviation value (SD) of the % viability should be ≤ 18.

INTERPRETATION OF RESULTS
According to EU classification, the irritancy potential of test items is predicted for distinguishing between R38 skin irritating and no-label (non-skin irritating) test substances OECD TG 404 & Method B.4 of Annex V to Directive 67/548/EEC. In the present study, the irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance. The test substance is considered to be irritant to skin (R38), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The test item was applied in its original form, no formulation was required.
The epidermal surface was first moistened with 10 μl deionised water and then 10 mg of the test item was applied evenly onto the skin. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.

Duration of treatment / exposure:

The plates with the treated epidermis units werei ncubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

Duration of post-treatment incubation (if applicable):

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.

Number of replicates:

Three replicates.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

> 50

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item showed reduced cell viability in comparison to the negative control (mean value: 67 %). However all obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin.

MTT REDUCTION
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.

OD EVALUATION
As the test item has an intrinsic colour (brown), one additional chemical-treated tissue was used for the non-specific OD evaluation. The OD (measured at 570 nm) of this tissue was determined as 0.024. The Non Specific Colour % (NSC %) was calculated as 3.0 %.
Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.819, the positive control result showed 6 % 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.

Any other information on results incl. tables

OD values and viability percentages of the test item

	Optical Density (OD)		Viability (%)
Test item	1	0.593	72
	2	0.629	77
	3	0.428	52
	Mean	0.550	67
	standard deviation (SD)		13.07

OD values and NSC % of additional control

	Optical Density (OD)		Viability (%)
test item treated tissues without MTT incubation	1	0.024	3.0

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells.

Controls	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.764	93
	2	0.860	105
	3	0.833	102
	Mean	0.819	100
	standard deviation (SD)		6.02
Positive Control: SDS (5 % aq.)	1	0.067	8
	2	0.045	6
	3	0.037	5
	Mean	0.050	6
	standard deviation (SD)		1.91

Applicant's summary and conclusion

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Not irritating.

Executive summary:

The irritation potential of test item has been assayed by measurement of its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. The experiment was conducted following the procedures outlined into the OECD guideline 439. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. 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 precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. The test item has an intrinsic colour (orange), one additional chemical-treated tissue was used for the non-specific OD evaluation. SDS (5 % aq.) and PBS treated epidermis were used as positive and negative controls respectively (three units / control). For each treated tissue viability was expressed as a percentage relative to negative control. The test item 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. The test item showed reduced cell viability in comparison to the negative control (mean value: 67 %). However all obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Conclusion

In the EPISKIN model, test item resulted to be non Irritant (NI) [UN GHS: No Category].