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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:
15 March 2017 - 31 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 2009
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
erature
Expiry date: 20 September 2017

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Episkin Laboratories, Lyon, France.
Details on animal used as source of test system:
The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin Small Model (0.38cm²)
- Tissue batch number: 17-EKIN-012
- Date of initiation of testing: 22 March 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: +37°C
- Temperature of post-treatment incubation: +37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 12 times with 2 mL D-PBS 1X
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology, Quality controls and Biological Safety verified by the test system supplier.
All information available in the test system certificate of analysis.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): EpiSkin tissues are placed in an incubator (set at +37°C, 5% CO2) for 48 hours (± 1 hour). At the end of the incubation period, the tissues will are dried before being placed in a freezer (-20°C) for at least one night.
- N. of replicates : 3
- Method of calculation used: the percentage of OD due to this non-specific MTT reduction is calculated and subtracted before calculation of the “true” viability percentages.
Non-Specific MTT reduction (NSMTT) calculation = [(mean cODTIK – mean cODUK) / mean cODNC] x 100
where cOD Untreated Killed (UK) tissues = ODUK – mean ODblank and cOD Test Item treated Killed (TIK) tissues = ODTIK – mean ODblank
If the NSMTT is > 30% relative to the negative control OD, additional steps may be undertaken (at additional cost; will be detailed in a study plan amendment) or the chemical will be considered as incompatible with the test.
If the NSMTT is 0% = NSMTT = 30% relative to the negative control OD, the corrected OD value of the test item-treated water-killed tissues may be subtracted from the mean cOD of the test item-treated living tissues to obtain the true amount of MTT reduction (i.e. the MTT reduction produced by metabolic conversion only).


NUMBER OF INDEPENDENT EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL:
- The test substance is considered to be irritant to skin if the mean viability obtained for the test substance is less than or equal to 50%, or if the mean viability os greater than 50% but the mean IL1a release exceeds 60 pg/mL
- The test substance is considered to be non-irritant if the mean viability value obtained for the test substance is less than 50% together with a mean IL1a below or equal to 60 pg/mL.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL (± 2 µL) of neat test item
Duration of treatment / exposure:
15 minutes (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Test system

Type of coverage:
other: not applicable (in vitro)
Preparation of test site:
other: not applicable (in vitro)
Vehicle:
unchanged (no vehicle)
Details on study design:
Preliminary tests
Test for direct MTT reduction with the test item
As a test substance may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test.
To identify any test item interference with the MTT endpoint, the following preliminary test was performed:
- 10 µL of the test item were added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 10 µL of water for injections to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 5 minutes).
Then the colour of the solutions obtained was evaluated.

Test for the detection of the colouring potential of the test item
The intrinsic colour or the ability of the test item to become coloured in contact with water (simulating a tissue humid environment) was evaluated by adding 10 µL of test item to 90 µL of water for injections in a transparent recipient. After 1 hour (± 3 minutes) of mixing, the presence and intensity of the colouration was evaluated.

Main test
One 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.

Pre-incubation of the tissues on their day of arrival (Day 0)
A volume of 2 mL of pre-warmed (at +37°C) maintenance medium was added to 3 wells on a 12 well plate (one plate per item).
Each EpiskinTM tissue was then transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at +37°C, 5% CO2 in a humidified incubator for at least 24 hours.
Treatment of tissues (Day 1)
A volume of 2 mL of pre-warmed maintenance medium was added into 3 wells on the 12 well plate for each item, respectively.
Each test or control item was then applied on three tissues for an exposure period of 15 minutes (± 1 minute) at room temperature and according to quantities defined in § Test item, positive and negative controls administrations. The items were applied topically to the corresponding tissues and gently spread over the epidermal surfaces to ensure uniform covering of the tissues. The tissues were processed (treatment and rinsing) in the same order and at regular time-intervals to ensure each tissue received an equal exposure period.

For the positive control only, the SDS solution was re-spread after 7 minutes (± 1 minute) contact time with a curved spatula to improve the distribution of the SDS for the remainder of the contact period.

Rinsing of tissues and incubation for 42 hours (Day 1)
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items.
The rinsed tissues were transferred to the second column of three wells containing 2 mL of maintenance medium in each well and the plates were incubated at +37°C, 5% CO2 in a humidified incubator for 42 hours.

MTT viability assay (Day 3)
Following the 42 hours incubation period, each 12-well plate was placed for 15 minutes ± 2 minutes on a plate shaker to homogenize the released inflammatory mediators in the maintenance medium. Then, 1.6 mL of incubation medium from beneath each tissue were retained in pre-labeled micro tubes and stored at -20°C until possible quantification for inflammatory mediators.

Two milliliters of a freshly prepared MTT solution (0.3 mg/mL) were added into 3 wells on each 12-well plate. The tissues were then transferred to the MTT filled wells and incubated for 3 hours at +37°C, 5% CO2 in a humidified incubator.

At the end of the MTT incubation period, a total biopsy of the epidermis was made by using the Episkin™ biopsy punch. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. The epidermis was separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into micro tubes and incubated with 500 µL of acidic isopropanol. Then, to extract the formazan (reduced MTT) from the MTT-loaded tissues, each tube was stored after vortexing at +5°C and protected from light until Day 6 of the experiment.

Optical Density measurements (Day 6)
At the end of the formazan extraction period, each tube was vortexed until the solution colour became homogenous.
Each tube was used to fill two wells of a 96-well plate with 200 µL of extract per well. One 96-well plate was used for the negative and positive controls (placed at opposite ends of the plate), and a separate 96-well plate was used for the test item.

For each 96-well plate, the average Optical Density value (OD) of six wells containing 200 µL of acidified isopropanol only was used as the blank.

The OD was measured at 570 nm using a plate reader.

Quantification of IL-1a by ELISA.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
15 min
Run / experiment:
Mean
Value:
78
Vehicle controls valid:
not examined
Negative controls valid:
yes
Remarks:
100%
Positive controls valid:
yes
Remarks:
5%
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: IL1a release
Remarks:
(pg/mL)
Run / experiment:
Main
Value:
104
Vehicle controls valid:
not examined
Negative controls valid:
yes
Remarks:
11.7
Positive controls valid:
not applicable
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Evaluation of the colouration of tissues at the end of the MTT incubation period
The qualitative evaluation of the MTT staining was performed with the naked eye.
All test item-treated tissues appeared blue which was considered indicative for viable tissues.

Evaluation of the MTT results
All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the study was considered to be valid.

Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 78% with a standard deviation of 7% as assessed by the MTT assay (Table 2).
As the mean viability was > 50% after the MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA.
The mean IL-1a concentration for test item-treated tissues was 104 pg/mL (Appendix 2). Due to this value being above 60 pg/mL, the results met the criteria for an in vitro classification as irritant to skin.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean cOD of the three negative controls should be = 0.6 and the Standard Deviation (SD) value of the % viability should be = 18%,
- Acceptance criteria met for positive control: the relative mean viability of the positive control should be = 40% of the relative mean viability of the negative control and the SD value of the % viability should be = 18%.

Any other information on results incl. tables

 

 

 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Category 1 (H314) or Category 2 (H315)
Conclusions:
The test item is considered to be irritant to skin; however, it is not possible to discriminate the classification between Category 1 (H314) and Category 2 (H315) (Regulation (EC) No. 1272/2008).
Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the EpiskinTM reconstructed human epidermis model. The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). In addition, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 78% with a standard deviation of 7%. As the mean viability was > 50% after the MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The mean IL-1a concentration for test item-treated tissues was 104 pg/mL. Due tothis value being above the limit of 60 pg/mL, the results met the criteria for an in vitro classification as irritant to skin.

Under the experimental conditions of this study, the test item is considered to be irritant to skin; however, it is not possible to discriminate the classification between Category 1 (H314) and Category 2 (H315) (Regulation (EC) No. 1272/2008).