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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:
23 March 2016 to 25 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Benzaldehyde, hydroxy-, polymer with phenol
Cas Number:
106466-55-1
Molecular formula:
(C7H6O2.C6H6O)x
IUPAC Name:
Benzaldehyde, hydroxy-, polymer with phenol
Test material form:
solid: flakes
Details on test material:
- Appearance: Red – reddish brown solid flake
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH %)
Specific details on test material used for the study:
- The test material was applied in its original form, no formulation was required (although the test item was ground to a fine powder).

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN™ (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ (SM)
- Tissue batch number(s): 16-EKIN-012 and 15-EKIN-045 (for killed epidermis)
- Expiry date: 28 March 2016 and 16 November 2015 (for killed epidermis)
- The EPISKIN™ (SM) kit was kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.

KIT RECEPTION
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- 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 colour change is irreversible, independent of the length of the period above 40 °C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

INDICATOR FOR POTENTIAL FALSE VIABILITY
- Check-method for possible direct MTT reduction with test material: Approximately 10 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded: test materials which do not react with MTT: yellow and test materials reacting with MTT: blue or purple. After three hours incubation, purplish colour of the mixture was detected in the test tube. Thus, the test material reacted with MTT and therefore the use of additional controls was necessary.
- Check-method to detect the colouring potential of test-materials: Prior to treatment, the test material was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test materials had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test materials to stain the epidermis by using additional control tissues.
The test material was showed being an MTT-interacting substance and the test material had an intrinsic colour so a third set of controls were necessary. The test material may bind to both living and killed tissues and therefore the NSMTT control may not only correct for potential direct MTT reduction by the test chemical, but also for colour interference arising from the binding of the test chemical to killed tissues. This could lead to a double correction for colour interference since the NSCliving control already corrects for colour interference arising from the binding of the test chemical to living tissues. To avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled) was needed.
- Therefore, in addition to the normal procedure, two additional test material-treated living tissues and two additional test material-treated killed tissues were used for the non specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.
- Process for test materials with both a direct MTT reduction with test material, and dyes and chemicals able to colour the tissue (and possibly affecting the absorption at the measurement wavelength): The test material was identified as producing both direct MTT reduction and colour interference was also require a third set of controls. This is usually the case with darkly coloured test chemicals interfering with the MTT assay (e.g., blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT. These test chemicals may bind to both living and killed tissues and therefore the NSMTT control may not only correct for potential direct MTT reduction by the test chemical, but also for colour interference arising from the binding of the test chemical to killed tissues. This could lead to a double correction for colour interference since the NSCliving control already corrects for colour interference arising from the binding of the test chemical to living tissues. To avoid a possible double correction for colour interference, a third set of control for non-specific colour in killed tissues (NSCkilled) was performed.

PRE-INCUBATION (DAY [-1])
- The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was 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, in a >95 % humidified atmosphere.

APPLICATION (DAY 0)
- Test Material: As the test material was solid, first an appropriate amount (10 μL) of distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 10 mg of the test material was applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
- Negative and positive controls: 50 μL of negative control (PBS) or positive control (5 % (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
- The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.9 - 26.7 °C).
- As the test material was showed being an MTT-interacting substance, in addition to the normal procedure, two test material treated killed epidermis and two negative control treated killed epidermis was used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues. The same treatment steps were followed in case of the killed tissues as in case of the living tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (23.9 - 26.7 °C)
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the 15 minutes incubation time, the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The test material stuck on surface of the epidermis additional rinsing was used. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the 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 AND FORMAZAN EXTRACTION (DAY 2)
- After the 42 hours incubation, all EPISKIN™ (SM) units (except of two colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). 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.
- After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved 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 containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate). The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
- Following the formazan extraction, 2 × 200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

NUMBER OF REPLICATE TISSUES: 3

VALIDITY OF THE TEST
- 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 values should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0-40 % and the standard deviation value (SD) of the % viability values should be ≤ 18.
- The SD calculated from individual % tissue viability values of the three test material treated replicates should be <18.
- The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

INTERPRETATION OF TEST RESULTS
- The irritation potential of test materials can be classified according to the United Nations Globally Harmonised System of Classification and Labelling of Chemicals [10,11], and a similar system is used in CLP. In the present study, the irritancy potential of test substances is predicted by the mean tissue viability of tissues exposed to the test material. The test material considered to be irritant to skin (Category 2), 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 prediction model (PM) is described below:
Mean tissue viability % is ≤ 50 % = Category 2
Mean tissue viability % is > 50 % = No Category
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 5 % (w/v)
Duration of treatment / exposure:
15 (± 0.5) minutes
Duration of post-treatment incubation (if applicable):
42 (± 1) hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
14.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ADDITIONAL CONTROLS
- As the test material was coloured, two additional test material-treated tissues were used for the non specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.049, Non Specific Colour % was calculated as 6.9 %. This value was above 5 %, therefore additional data calculation was necessary.
- As colour change (purplish) was observed after three hours of incubation of the test material in MTT working solution, thus the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Based on these observed mean OD (0.054), the calculated NSMTT is 7.5 %. This was considered to be significant, thus additional data calculation was necessary.
- As the test material was showed being an MTT-interacting substance and the test material had an intrinsic colour, two additional test material-treated killed tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.067, Non Specific Colour % (NSCkilled%) was calculated as 9.5 %. Because the NSCliving% was used, therefore correction with NSCkilled% was necessary.

VIABILITY RESULTS
- The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The OD values for the test material treated skin samples showed 14.9 % relative viability.

VALIDITY OF THE TEST
- After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- The mean OD value of the three negative control tissues was in the recommended range (0.710). Standard deviation of the viability results for negative control samples was 6.3.
- The positive control treated tissues showed 9.0 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 3.5.
- The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 1.3.
- The mean OD value of the blank samples (acidified isopropanol) was 0.046.
- All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Treatment

Optical Density (OD)

TODTT

Viability (% RV)

 

Measured

Blank Corrected

Negative Control (PBS)

1

0.755

0.708

-

99.7

2

0.713

0.667

-

93.9

3

0.802

0.756

-

106.4

Mean

-

0.710

-

100.0

Positive Control (5 % (w/v) SDS Solution)

1

0.095

0.049

-

6.9

2

0.139

0.093

-

13.0

3

0.098

0.051

-

7.2

Mean

-

0.064

-

9.0

Test Material

1

0.187

0.140

0.105

14.8

2

0.197

0.150

0.115

16.2

3

0.179

0.132

0.097

13.6

Mean

 

0.141

0.106

14.9

Mean blank value was 0.046.

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

TODTT: The measured values were corrected for non-specific MTT reduction (by subtracting the NSMTT value of 0.054) and for non-specific colour reduction (by subtracting the NSCliving value of 0.049 and by added the NSCkilled value of 0.067).

Applicant's summary and conclusion

Interpretation of results:
other: EU Criteria: Category 2, Causes skin irritation (H315)
Conclusions:
Under the conditions of this study, the test material is irritating to the skin.
Executive summary:

An in vitro skin irritation test of the test material was performed in a reconstructed human epidermis model. EPISKIN (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test material was evaluated according to the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions.

Disks of EPISKIN (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The 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 an incubator with 5 % CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test material. The possible MTT interaction potential of the test material was examined using two additional test material treated and two negative control treated killed epidermis units. Furthermore to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. 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 material is considered to be irritant to skin.

Following exposure to the test material, the mean cell viability was 14.9 % (after adjustment for non-specific MTT reduction, NSCliving and NSCkilled) compared to the negative control. This is below the threshold of 50 %, therefore the test material was considered as being irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of this study, the test material is irritating to the skin.

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