Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 November 2017 to 10 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”,
Version / remarks:
Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, No. 431, (29 July 2016) “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Chemical name: α,α’-Dichloro-p-xylene
CAS number: 623-25-6
Batch/Lot Number: EWP6O
Description: White-Pale yellow green, Solid
Purity*: >98.0% (GC)
Expiry date: 17 August 2018 / 30 October 2019
Storage conditions: Controlled room temperature (15-25 °C, ≤ 70 RH%), protected from humidity (tight closed container), protected from light
Safety precautions: Enhanced safety precautions were applied considering the supplied safety data sheet to assure personnel health and safety. Fatal if inhaled!
Specific details on test material used for the study:
No further details specified in the study report.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not applicable
Source strain:
other: Not applicable
Details on animal used as source of test system:
Human Skin
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-045, Expiry Date: 13 November 2017) 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., 1994). Its use for skin corrosivity testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKINTM(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 a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkinTM(SM) Test Kits used in the present study).
Justification for test system used:
The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
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)
The Assay 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, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.

Rinsing (Day 0)
After the incubation time (4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT test (Day 0)
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere for 3 hours (±15 minutes), protected from light.

Formazan extraction (Day 0)
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure 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 overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Cell viability measurements (Day 1)
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.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
- 20 mg of test item was applied evenly to the epidermal surface of each of two test item treated skin units and each additional control skin units and then 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (22.3-24.1°C) covered with the plate lids.
Duration of post-treatment incubation (if applicable):
Plate incubated at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere for 3 hours (±15 minutes), protected from light.
Number of replicates:
In this assay, two replicates per test item were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
107.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ADDITIONAL CONTROLS
As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.003, Non Specific Colour% (NSCliving%) was calculated as 0.3%. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.
As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

VIABILITY RESULTS
The mean OD value for the test item treated skin samples showed an 107.7% relative viability compared to the negative control.

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 two negative control tissues was in the recommended range (1.065).
The two positive control treated tissues showed 1.0% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 4.3%.
The difference of viability between the two negative control tissue samples in the MTT assay was 9.2%.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues

Additional control

Optical Density (OD)

NSC% (living)

 

Measured

Blank corrected

Treated with

α,α’-Dichloro-p-xylene

1

0.054

0.007

0.3

2

0.046

0.000

Mean

--

0.003

Notes:

1. Mean blank value was 0.046

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

 

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

Substance

Optical Density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative Control:Physiological saline

(0.9% (w/v) NaCl)

1

1.063

1.016

95.4

2

1.161

1.114

104.6

Mean

--

1.065

100.0

Positive Control:

Glacial acetic acid

1

0.061

0.015

1.4

2

0.052

0.006

0.5

Mean

--

0.010

1.0

Test Item:

α,α’-Dichloro-p-xylene

1

1.219

1.172

110.0

2

1.169

1.123

105.4

Mean

--

1.147

107.7

Notes:

1. Mean blank value was 0.046

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

 

HISTORICAL CONTROL DATA

(updated 18 January 2018)

 

Negative control

(physiological saline)

Positive control

(Glacial acetic acid)

Mean optical density (OD)

0.864

0.014

Standard deviation

0.149

0.010

Minimum optical density (OD)

0.590

0.000

Maximum optical density (OD)

1.516

0.051

Number of cases

130

130

Note: All optical density (OD) values measured are background corrected values (measured at 570±30 nm).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure with α,α’-Dichloro-p-xylene, the mean cell viability was 107.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN™(SM) model test with α,α’-Dichloro-p-xylene (Batch number: EWP6O), the results indicate that the test item is non-corrosive to the skin.
Executive summary:

An in vitro skin corrosivity test of α,α’-Dichloro-p-xylene test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive 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 corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

 

Disks of EPISKINTM(SM) (two units) were treated with α,α’-Dichloro-p-xylene test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

 

Following exposure with α,α’-Dichloro-p-xylene, the mean cell viability was 107.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore, the study was considered to be valid.

 

In conclusion, in this in vitro EPISKIN™(SM) model test with α,α’-Dichloro-p-xylene (Batch number: EWP6O), the results indicate that the test item is non-corrosive to the skin.