Registration Dossier

Administrative data

Description of key information

Skin corrosion - In vitro

α,α’-Dichloro-p-xylene is non-corrosive to the skin.

Skin Irritation - In vitro

α,α’-Dichloro-p-xylene is irritant to skin, UN GHS Classification: Category 2.

Eye irritation - In vitro

α,α’-Dichloro-p-xylene, the test item was non-irritant, UN GHS Classification: No Category.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
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)
Specific details on test material used for the study:
No further details specified in the study report.
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.
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.

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).

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.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January 2018 to 02 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
OECD Guidelines No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (28 July 2015)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No 761/2009 of 23 July 2009, ANNEX III, B.46., “In Vitro Skin Irritation Reconstructed Human Epidermis Model Test”, amended by Commission Regulation (EU) No 640/2012 of 6 July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No further details specified in the study report.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: three-dimensional human epidermis model.
Source strain:
other: Not applicable
Details on animal used as source of test system:
EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-005, Expiry Date: 05 February 2018) is a three-dimensional human epidermis model. Adult humanderived 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 irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
Justification for test system used:
The EPISKINTM (SM) model has been validated for irritation testing in an international validation study [10] 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:
INDICATOR FOR POTENTIAL FALSE VIABILITY
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.

Check-method for possible direct MTT reduction with test item
10 mg of powdered test item 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 items which do not react with MTT: yellow
-Test items reacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube.
Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

Check-method to detect the colouring potential of test-items
Prior to treatment, the test item was evaluated for their intrinsic colour or ability to become coloured in contact with water* and/or extracting solution (e.g. acidified isopropanol) (simulating a tissue humid environment).
*Water is the environment during exposure.
Note: The Non-Specific Colour % (NSCliving %) was determined in study 17/241-039B, In Vitro Skin Corrosivity Test in the EPISKINTM (SM) Model. This value (0.3%) was below 5% and additional data calculation was not necessary, therefore check-method for colouring potential of test-item the use of additional controls for the non-specific OD evaluation was not necessary in this study.

PERFORMANCE OF THE STUDY
Procedures were performed under aseptic conditions (in sterile hood using sterile equipment).

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 and rinsing (Day 0)
Test Item
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of the powdered test item was 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).
Note: The negative and positive controls were also part of a concurrent study (Citoxlab study code: 17/384-043B; 17/397-043B; 17/373-043B) performed in the same experimental period using the same batch of chemicals and same batch of skin units.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature (22.5-24.9°C).
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% humidified atmosphere.

MTT test (Day 2)
After the 42 hours incubation, all EPISKINTM (SM) units were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

Formazan extraction (Day 2)
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 (Day 2)
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:
10 mg of the powdered test item was applied evenly to the epidermal surface.
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit.
Duration of treatment / exposure:
15 minutes.
Duration of post-treatment incubation (if applicable):
Incubated for 42 hours (± 1h) at 37°C in an incubator
Number of replicates:
In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
38.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ADDITIONAL CONTROLS
As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
Note: The Non-Specific Colour % (NSCliving %) was determined in study 17/241-039B, In Vitro Skin Corrosivity Test in the EPISKINTM (SM) Model. This value (0.3%) was below 5% and additional data calculation was not necessary, therefore check-method for colouring potential of test-item the use of additional controls for the non-specific OD evaluation was not necessary in this study.

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 2. The mean OD values for the test item treated skin samples showed 38.8% 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 three negative control tissues was in the recommended range (0.918). Standard deviation of the viability results for negative control samples was 5.4%.
The positive control treated tissues showed 3.7% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.0%.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 0.8%.
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.

Following exposure with α,α’-Dichloro-p-xylene, the mean cell viability was 38.8% compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

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

Substance

Optical Density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative Control:

Phosphate buffered saline

1

2

3

0.928

1.021

0.945

0.881

0.974

0.898

96.0

106.1

97.9

Mean

--

0.918

100.0

Positive Control:

5% (w/v) SDS solution

1

2

3

0.076

0.075

0.092

0.029

0.028

0.045

3.2

3.1

4.9

Mean

--

0.034

3.7

Test Item:

α,α'-Dichloro-p-xylene

1

2

3

0.395

0.406

0.408

0.348

0.359

0.361

37.9

39.2

39.4

Mean

--

0.356

38.8

Notes:

1. Mean blank value was 0.047

2. Optical density vales 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

(PBS)

Positive control

(5% (w/v/) SDS solution)

Mean optical density (OD)

0.788

0.065

Standard deviation

0.129

0.041

Minimum optical density (OD)

0.573

0.019

Maximum optical density (OD)

1.362

0.354

Number of cases

251

246

PBS: Phosphate buffered saline

SDS: Sodium dodecyl sulphate

OD: Optical density (absorbance)

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

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Following exposure with α,α’-Dichloro-p-xylene, the mean cell viability was 38.8% compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKINTM (SM) model test with α,α’-Dichloro-p-xylene (Batch number: EWP6O), the results indicate that the test item is irritant to skin, UN GHS Classification: Category 2.
Executive summary:

An in vitro skin irritation 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 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 item was evaluated according to the OECD No. 439 guideline.

 

Disks of EPISKINTM(SM) (three units) were treated with the powdered 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, in a >95% humidified atmosphere. 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, in a >95% humidified atmosphere, 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 α,α’-Dichloro-p-xylene, the mean cell viability was 38.8% compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

 

Based on the results of the corrosivity test (performed by Citoxlab Hungary Ltd., study code: 17/241-039B) the test item is non-corrosive.

 

In conclusion, in this in vitro EPISKINTM(SM) model test with α,α’-Dichloro-p-xylene (Batch number: EWP6O), the results indicate that the test item is irritant to skin, UN GHS Classification: Category 2.

Endpoint:
skin irritation / corrosion, other
Remarks:
QSAR assessment
Type of information:
(Q)SAR
Remarks:
Predicted using OECD QSAR Toolbox & DEREK Nexus
Adequacy of study:
key study
Study period:
25 January 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
QSAR prediction
Justification for type of information:
The study is currently ongoing.  This  submisison allows co-registrants to register for the deadline and the lead dossier will be spntaneously updated as soon as the result is available.
Principles of method if other than guideline:
Data is QSAR data but the study is currently ongoing.  This  submisison allows co-registrants to register for the deadline and the lead dossier will be spntaneously updated as soon as the result is available.
Test system:
other:
Remarks:
QSAR Assessment
Irritation / corrosion parameter:
other:
Remarks:
QSAR assessment
Run / experiment:
DEREK nexus
Remarks on result:
positive indication of irritation
Remarks:
QSAR prediction
Irritation parameter:
other:
Remarks:
QSAR prediction
Basis:
other:
Remarks:
QSAR prediction
Time point:
other: QSAR prediction
Reversibility:
other:
Remarks:
QSAR prediction
Remarks on result:
positive indication of irritation
Remarks:
QSAR prediction
Irritant / corrosive response data:
Predicted positive for skin irritation
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Predicted as potential skin irritant
Conclusions:
Predicted positive for skin irritation following in-silico assessments using OECD Tool Box Rules by BfR and Derek Nexus
Executive summary:

The study is currently ongoing.  This  submisison allows co-registrants to register for the deadline and the lead dossier will be spntaneously updated as soon as the result is available. The skin sensitisation has  been  assessed  by  assessment  of  the  structure  for  structural  alerts

compared  to  known  alerts contained in the OECD Tool Box Rules by BfRD EREK Nexus database. On the basis of the structure the substance is considered as a skin irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 November 2017 - 09 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
OECD Guidelines for Guidelines for Testing of Chemicals No.438 (09 October 2017) “Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage”
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 method B.48 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) “Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants”
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
The test item of a suitable chemical purity was supplied by the Sponsor. All precautions required in the handling and disposal of the test item were outlined by the Sponsor. These documents are part of the raw data. Test item was identified on the basis of the information provided by the Sponsor by the Pharmacy of Citoxlab Hungary Ltd.

Test item solubility
The solubility of the test item in physiological saline was tested prior to the experiment (30 mg test material in 1 mL physiological saline). The test item did not dissolve in physiological saline.

Formulation
The test item was applied in its original form, although it was powdered.
Species:
chicken
Strain:
other:
Remarks:
COBB 500
Details on test animals or tissues and environmental conditions:
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Citoxlab Hungary Ltd. at ambient temperature at the earliest convenience.After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Citoxlab Hungary Ltd. and processed within 2 hours of collection in each experiment.

Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluoresceintreated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was applied in its original form, although it was powdered.
Duration of treatment / exposure:
The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. Slight corneal thickness changes (0.0 - 1.6 %) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Treatment
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In each experiment, 30 mg of the powdered test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea. In each experiment negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole. One eye was treated with physiological saline, three eyes with the test item and another three eyes with powdered imidazole in each experiment.
Observation period (in vivo):
Observation and assessment of corneal effects The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

Morphological effects
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
Duration of post- treatment incubation (in vitro):
Test item removal
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible. Additional gentle rinsing with 20 mL saline was performed after treatment and at each time point when the test item or positive control material remaining on the cornea was observed in each experiment.
Number of animals or in vitro replicates:
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.
Details on study design:
The Enucleated Eye Test with isolated eyes of chickens is a well validated and accepted in vitro test system. It has been recognised as a valuable alternative to the Draize eye irritation test, because it represents a test system nearest to the in vivo test, without the need to use live animals. It can also be used as a screening tool for corrosivity/severe irritancy to avoid unacceptable effects in vivo. In the Isolated Chicken Eye Test (ICET) the test compound is applied in one single dose onto the cornea of isolated eyes. Chicken heads are obtained from a veterinary-inspected, commercial slaughter-house, processing chickens for human consumption. This method can provide detailed information about the effects of test items on the cornea, and can be used to identify chemicals not requiring classification for eye irritation, or for serious eye damage and chemicals inducing serious eye damage as defined by the UN GHS (UN GHS non-classified or UN GHS Category 1). The test is described in OECD No. 438 and is approved by international regulatory agencies as a replacement for the identification of non-irritant, corrosives/severe irritants in the in vivo Rabbit Eye Assay (OECD No. 405).
Irritation parameter:
morphological effects
Value:
>= 1.1 - <= 1.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Value:
>= 0 - <= 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Value:
>= 0 - <= 0.17
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test item α,α’-Dichloro-p-xylene showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes with α,α’-Dichloro-p-xylene, the test item was non-irritant, UN GHS Classification: No Category.

MORPHOLOGICAL EFFECTS RESULTS
The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse in each experiment.
No other morphological effect was observed in the study.

TEST ITEM

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

1.1%

I

Mean maximum corneal swelling at up to 240 min

1.6%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.17

I

Other Observations

None

Overall ICE Class

3xI

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

1.1%

I

Mean maximum corneal swelling at up to 240 min

1.1%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

POSITIVE CONTROL

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

12.2%

III

Mean maximum corneal swelling at up to 240 min

28.2%

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other Observations

Imidazole was stuck on all cornea surfaces at 30 minutes after the post-treatment rinse.

The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Overall ICE Class

1xIII 2xIV

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

13.4%

III

Mean maximum corneal swelling at up to 240 min

27.3%

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other Observations

Imidazole was stuck on all cornea surfaces at 30 minutes after the post-treatment rinse.

The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Overall ICE Class

1xIII 2xIV

 

NEGATIVE CONTROL

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0%

I

Mean maximum corneal swelling at up to 240 min

0.0%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0%

I

Mean maximum corneal swelling at up to 240 min

0.0%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

TABLES OF INDIVIDUAL DATA

Table of individual data α,α’-Dichloro-p-xylene (Experiment I)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

11

61

61

0.0%

61

0.0%

61

0.0%

0.0%

62

1.6%

62

1.6%

62

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0

0.0

12

60

61

1.7%

61

0.0%

62

1.6%

1.6%

62

1.6%

62

1.6%

62

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0.5

0.5

13

61

61

0.0%

61

0.0%

62

1.6%

1.6%

62

1.6%

62

1.6%

62

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0

0.0

Mean values:

0.0%

 

1.1%

1.1%

 

1.6%

 

1.6%

 

1.6%

1.6%

 

 

 

 

 

 

0.00

 

 

0.17

 

Table of individual data α,α’-Dichloro-p-xylene (Experiment II)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

11

63

63

0.0%

63

0.0%

3.

0.0%

0.0%

64

1.6%

64

1.6%

64

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0

0.0

12

63

63

0.0%

63

0.0%

63

0.0%

0.0%

63

0.0%

63

0.0%

63

0.0%

0.0%

0

0

0

0

0

0

0.0

0.5

0.5

0.0

13

61

61

0.0%

61

0.0%

61

0.0%

0.0%

62

1.6%

62

1.6%

62

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0

0.0

Mean values:

0.0%

 

0.0%

0.0%

 

1.1%

 

1.1%

 

1.1%

1.1%

 

 

 

 

 

 

0.00

 

 

0.00

 

Table of individual data (Imidazole) (Experiment I)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

14

60

60

0.0%

64

6.7%

67

11.7%

11.7%

70

16.7%

74

23.3%

78

30.0%

30.0%

0

4

4

4

4

4

4.0

0

3

3.0

15

61

61

0.0%

65

6.6%

68

11.5%

11.5%

70

14.8%

73

19.7%

77

26.2%

26.%

0

4

4

4

4

4

4.0

0

3

3.0

16

60

60

0.0%

65

8.3%

68

13.3%

13.3%

71

18.3%

74

23.3%

77

28.3%

28.3%

0

4

4

4

4

4

4.0

0

3

3.0

Mean values:

7.2%

 

12.2%

12.2%

 

16.9%

 

22.1%

 

28.2%

28.2%

 

 

 

 

 

 

4.00

 

 

3.00

Note: Imidazole was stuck on all cornea surfaces at 30 minutes after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

 

Table of individual data (Imidazole) (Experiment II)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

14

62

62

0.0%

66

6.5%

70

12.9%

12.9%

73

17.7%

76

22.6%

79

27.4%

27.4%

0

4

4

4

4

4

4.0

0

3

3.0

15

63

63

0.0%

68

7.9%

71

12.7%

12.7%

74

17.5%

77

22.2%

79

25.4%

25.4%

0

4

4

4

4

4

4.0

0

3

3.0

16

62

62

0.0%

68

9.7%

71

14.5%

14.5%

73

17.7%

77

24.2%

80

29.0%

29.0%

0

4

4

4

4

4

4.0

0

3

3.0

Mean values:

8.0%

 

13.4%

13.4%

 

17.6%

 

23.0%

 

27.3%

27.3

 

 

 

 

 

 

4.00

 

 

3.00

Note: Imidazole was stuck on all cornea surfaces at 30 minutes after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

 

Table of individual data (Physiological saline) (Experiment I)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

17

62

62

0.0%

62

0.0%

62

0.0%

0.0%

62

0.0%

62

0.0%

62

0.0%

0.0%

0

0

0

0

0

0

0.00

0

0

0.00

 

Table of individual data (Physiological saline) (Experiment II)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

17

63

63

0.0%

63

0.0%

63

0.0%

0.0%

63

0.0%

63

0.0%

63

0.0%

0.0%

0

0

0

0

0

0

0.00

0

0

0.00

 

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these in vitro eye irritation in the isolated chicken eyes tests with α,α’-Dichloro-p-xylene, the test item was non-irritant, UN GHS Classification: No Category.
Executive summary:

SUMMARY

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (09 October 2017).

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg powdered test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined in each experiment. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. Thus, the study was considered to be valid.

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No corneal opacity change was noted on test item treated eyes. No significant fluorescein retention change (severity 0.5) was noted on one test item treated eye and no fluorescein retention change was noted on two test item treated eyes. No other corneal effect was observed.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No corneal opacity change was noted on test item treated eyes. No fluorescein retention change was noted on test item treated eyes. No other corneal effect was observed.

Based on these in vitro eye irritation in the isolated chicken eyes tests with α,α’-Dichloro-p-xylene, the test item was non-irritant, UN GHS Classification: No Category.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion - In vitro

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.

 

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.

Skin Irritation - In vitro

Disks of EPISKINTM (SM) (three units) were treated with the powdered 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, in a >95% humidified atmosphere. 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, in a >95% humidified atmosphere, 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 α,α’-Dichloro-p-xylene, the mean cell viability was 38.8% compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin.

Eye irritation - In vitro

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes.

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg powdered test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined in each experiment.

 

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No corneal opacity change was noted on test item treated eyes. No significant fluorescein retention change (severity 0.5) was noted on one test item treated eye and no fluorescein retention change was noted on two test item treated eyes. No other corneal effect was observed.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No corneal opacity change was noted on test item treated eyes. No fluorescein retention change was noted on test item treated eyes. No other corneal effect was observed.

 

Based on these in vitro eye irritation in the isolated chicken eyes tests with α,α’-Dichloro-p-xylene, the test item was non-irritant.

Justification for classification or non-classification

In the in vitro EPISKINTM (SM) model test with α,α’-Dichloro-p-xylene (Batch number: EWP6O), the results indicate that the test item is irritant to skin, UN GHS Classification: Category 2.

Based on the in vitro eye irritation in the isolated chicken eyes tests with α,α’-Dichloro-p-xylene, the test item was non-irritant, UN GHS Classification: No Category.