Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 6, 2016 to December 16, 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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
September 22, 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes
Cell source:
other: three-dimensional human epidermis model
Justification for test system used:
The EPISKIN 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: three-dimensional human epidermis model
- Tissue batch number(s): 16-EKIN-042
- Expiration date: 24 October 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37°C
- Temperature used during treatment / exposure: 15 minutes at room temperature (23.0 - 24.7°C)
- Temperature of post-treatment incubation: 37°C

APPLICATION
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min).

REMOVAL OF TEST MATERIAL
After the 15 minutes incubation time, the EPISKINTM(SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. 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 in a 95% humified atmosphere.

MTT TEST
After the 42 hours incubation, all EPISKIN units (except of two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, the transferred EPISKIN units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light.

FORMAZAN EXTRACTION
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.

CELL VIABILITY MEASUREMENTS
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

PREDICTION MODEL / DECISION CRITERIA
In the present study, the irritancy potential of test substances is predicted by the mean tissue viability of tissues exposed to the test item. The test item 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.
Mean tissue viability % is ≤ 50%: Category 2 or Category 1
Mean tissue viability % is > 50%: Non-Irritant

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 item treated replicates should be ≤ 18.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST ITEM
10 μL of test item were applied evenly to the epidermal surface. If necessary, the test item 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).
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 tissues

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
107.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ADDITIONAL CONTROLS
As no colour change was observed after three hours of incubation of the test item in MTT working solution, thus the test item did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.
As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.012, Non-specific Colour % was calculated as 1.6%. This value was below 5%, therefore additional data calculation was not 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 below (Any other information on results incl. tables). The OD values for the test item treated skin samples showed 107.6% 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 (0.707). Standard deviation of the viability results for negative control samples was 2.8.
The positive control treated tissues showed 6.1% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.7.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 7.6.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
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

Substance

Optical Density (OD)

Viability

 

Measured

Blank corrected

(% RV)

Negative Control :

1

0.731

0.684

96.7

Phosphate buffered saline

2

0.764

0.717

101.4

 

3

0.767

0.720

101.9

 

mean

-

0.707

100.0

Positive Control :

1

0.077

0.030

4.2

5% (w/v) SDS* solution

2

0.101

0.054

7.6

 

3

0.093

0.043

6.5

 

mean

-

0.043

6.1

Test Item :

1

0.751

0.704

99.6

6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol

2

0.815

0.768

108.6

 

3

0.858

0.811

114.7

 

mean

-

0.761

107.6

* SDS: Sodium Dodecyl Sulphate

Notes:

1. Mean blank value was 0.047.

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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the in vitro EPISKIN test, 6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol is non-irritant to skin.
Executive summary:

An in vitro skin irritation test of 6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol test item was performed in a reconstructed human epidermis model EPISKIN (SM) according to the OECD No. 439 guideline.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item 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 atin 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). 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 item is considered to be irritant to skin.

Following exposure with 6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol, the mean cell viability was 107.6% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.