Registration Dossier

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Description of key information

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:
From March 23, 2017 to March 24, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
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.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EPISKINTM(SM)
Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-012, Expiry Date: 27 March 2017.
EPISKINTM(SM) 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.

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).
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 μl
Duration of treatment / exposure:
4 hours
Number of replicates:
Two replicates per test substance were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test substance was coloured, two additional test substance-treated tissues were used for the non specific OD evaluation.
Irritation / corrosion parameter:
% tissue viability
Value:
65.5
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked in each case. 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 (0.915).
- The two positive control treated tissues showed 0.8 % viability demonstrating the proper performance of the assay.
- The difference of viability between the two test substance-treated tissue samples in the MTT assay was 11.7 %.
- The difference of viability between the two negative control tissue samples in the MTT assay was 21.2 %.
- 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 relative viability % of the samples:

Substance

Optcal density (OD)

Viability (%)

Measured

Blank corrected

Negative control

Physiological saline

(0.9% (w/v) NaCl)

 

1.058

1.011

110.6

0.864

0.818

89.4

.

mean: 0.915

100

Positive control

Glacial acetic acid

0.059

0.013

1.4

0.048

0.001

0.1

 

mean: 0.007

0.8

Test substance

0.681

0.634

69.4

0.611

0.564

61.7

 

mean: 0.599

65.5

Mean blank value was 0.046.

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving %) of the additional control tissues:

Substance

Optcal density (OD)

NSCliving%

Measured

Blank corrected

Additional control

0.053

0.006

0.4

0.047

0.000

 

mean: 0.003

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the results of the in vitro EPISKIN™(SM) model test with the test substance indicate that the test substance is non corrosive to the skin.
Executive summary:

A study was conducted to determine the skin corrosion potential of the test substance using reconstructed human epidermis model EPISKINTM(SM) according to OECD Guideline 431 and EU-Method B.40-BIS, in compliance with GLP. Disks of EPISKINTM(SM) (two units) were treated with test substance and incubated for 4 hours at room temperature. Exposure of test substance 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 substance. 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 substance is considered to be corrosive to skin. The experiment met the validity criteria, therefore the study was considered to be valid. Following exposure with the test substance the mean cell viability was 65.5 % compared to the negative control. This is above the threshold of 35%, therefore the test substance was considered as being non-corrosive. Under the study conditions, the results of the in vitro EPISKIN™(SM) model test with the test substance indicate that the test substance is non corrosive to the skin (Orovecz, 2017).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 26, 2017 to April 28, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
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 irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
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)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
EPISKINTM (SM) is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. 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. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller C. et al., 2002, Mosmann T., 1983). The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. The % reduction in viability is used to predict the irritation potential of the test substance.
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-017, Expiry Date: 01 May 2017).
EPISKINTM (SM) 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 irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-008, Expiry Date: 27 February 2017) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37 °C in an incubator with 5 % CO2, in a >95 % humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to living tissues.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
20 µL of pre- warmed test substance (~37 °C) were applied evenly to the epidermal surface.If necessary, the test substance 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.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours incubation at 37 °C, 5 % CO2. Subsequent tissues incubation for 3 hours with MTT solution (at 37 °C, 5 % CO2 protected from light)
Number of replicates:
3 replicates for the treatment with the test substance and three units per negative and positive controls.
Additional controls to examine following effects:
- colour contribution (NSCliving) from the test substance
- the possible MTT interaction potential of the test substance
- non-specific colour in killed tissues (NSCkilled)
Irritation / corrosion parameter:
% tissue viability
Value:
6.2
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
As colour change (purple) was observed after three hours of incubation of the test substance in MTT working solution, thus the test substance might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Additional controls on killed epidermis were included in the study. Based on observed mean OD (-0.012), the calculated NSMTT% is 1.5 %, the OD values were negative so these values excluded from the NSMTT calculation.

As the test substance was coloured, two additional test substance-treated tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.023, Non Specific Colour % was calculated as 3.0 %. This value was below 5 %, therefore additional data calculation was not necessary.

As the test substance was showed being an MTT-interacting substance and the test substance had an intrinsic colour, two additional test substance-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 0.020, Non Specific Colour % (NSCkilled%) was calculated as 2.6 %. Because the NSCliving% was not used, therefore correction with NSCkilled% was not necessary.

Validity criteria:
- The mean OD value of the three negative control tissues was in the recommended range (0.776). Standard deviation of the viability results for negative control samples was 4.7.
- The positive control treated tissues showed 3.4 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.5.
- The standard deviation of viability values of the three test substance-treated tissue samples in the MTT assay was 1.3.
- 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.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the study conditions, following exposure with the test substance, the mean cell viability was 6.2 % compared to the negative control. This is below the threshold of 50 %, therefore the test substance was considered as being irritant to skin.
Executive summary:

A study was conducted to determine the skin irritation potential of the test substance using Reconstructed Human Epidermis (RHE) Test Method according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Three disks of EPISKINTM (SM were treated with the pre-warmed test substance and incubated for 15 minutes at room temperature. Exposure of the test substance 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 substance. The possible MTT interaction potential of the test substance was examined using two additional test substance 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. The experiment met the validity criteria, therefore the study was considered to be valid. Under the study conditions, following exposure with the test substance, the mean cell viability was 6.2 % compared to the negative control. This is below the threshold of 50 %, therefore the test substance was considered as being irritant to skin (Orovecz, 2017).

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:
From February 01 to February 07, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
other: Bos taurus
Details on test animals or tissues and environmental conditions:
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre_sur Dives, France.

Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).

Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.

Transport from Supplier to Citoxlab France: the eyes were immerged in containers filled with cooled buffered Hanks medium and placed into a cooling-box with a sufficient amount of ice packs to ensure cooling until arrival at Citoxlab France. Containers with smooth internal surfaces were used for the transport to avoid damage to the corneas. Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].

Preparation of the corneas:
Upon arrival at Citoxlab France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using Hanks’ Balanced Salt Solution (HBSS) in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swivelled in order to observe the fringe areas and any scratches directly under the light.

Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared. The corneas were used immediately.
Vehicle:
other: 0.9 % NaCl
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Test substance formulation:
Prior handling, a small portion of test substance was taken and placed at 37 °C until the liquid form was retrieved; i.e. in 10 minutes for the preliminary assay and 37 minutes for the main test. As the test substance was a surfactant, it was tested at the concentration of 10% (w/v) in the vehicle. The test substance formulation was a whitish emulsion. The test substance formulation was prepared within 4 hours of use and stored at room temperature until use.

Dosing:
A volume of 750 µL (± 8 µL) was applied on each cornea using the closed-chamber treatment method as follows: the test substance formulation was introduced into the anterior chamber of the corneal holder, through the dosing holes to cover the epithelial side of the cornea. The dosing holes were then sealed.
Duration of treatment / exposure:
10 minutes
Number of animals or in vitro replicates:
3
Details on study design:
Pre-incubation:
Both chambers of the corneal holder were filled to overflowing with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. The posterior chamber was always filled first to maintain the natural concave shape of the cornea.
After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C).

At the end of the pre-incubation period, the medium was removed from both chambers of the holder using a metal gavage tube attached to a vacuum pump to ensure complete evacuation. They were refilled with fresh cMEM without phenol red (previously heated to +32°C), starting with the posterior chamber and taking care that no air bubbles were present. The chambers were re-sealed and the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0.

Allocation of the corneas:
- The median value of the OPT0 values of all pre-incubated corneas (with OPT0 ≤ 7) was calculated,
- Three corneas with opacity values close to the median value were selected as vehicle control corneas,
- The remaining corneas were shared out between test substance and positive control-treated series using a manual distribution procedure.

Treatment of corneas:
The medium of the anterior chamber was removed and each substance was applied onto the epithelium of the cornea, test substance formulation, positive and vehicle controls application.
After application of the substance, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32°C (± 1°C), for the selected treatment time.

Rinsing of the corneas:
On completion of the treatment period, the test substance formulation was removed from the front opening of the anterior chamber (closed-chamber method) and the epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- the corneas were rinsed three times with pre-warmed cMEM containing phenol red. Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

The anterior chamber was refilled with fresh pre-warmed cMEM without phenol red. The front cover was replaced. Care was taken to make sure that no air bubbles were present within the holders.
Following the 10-minute treatment and the rinsing step, the holders were incubated horizontally (corneas placed vertically) for 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C). On completion of the 2-hour incubation period, the medium of both anterior and posterior chambers was renewed with pre-warmed cMEM (+32°C (± 1°C)), the second opacity measurement (OPT2) was then performed.

Opacity measurements:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.

Just before the first opacity measurement (i.e. OPT0), the opacitometer was calibrated using specific calibrators. Values obtained for each calibrator were as follows:
- calibrator No. 1: set to 75,
- calibrator No. 2: from 145 to 155,
- calibrator No. 3: from 218 to 232.

Just before the second opacity measurement (i.e. OPT2), the opacitometer was calibrated using the calibrator No. 1 set to 75.
Just after each opacity measurement (OPT0 and OPT2), the calibration of the opacitometer was checked by using the calibrator No. 1. The obtained value was between 73 and 77.
Corneal opacity was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left hand chamber.

Permeability determination:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution.

As the test substance was a surfactant, the concentration of the fluorescein solution was 4 mg/mL. Before use, the fluorescein solution was validated.

For each series of three corneas, a chronometer started from the fluorescein solution application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).

At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).

Macroscopic examination:
After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium.
Irritation parameter:
in vitro irritation score
Value:
3
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- No notable opaque spots or irregularities were observed on vehicle control corneas.
- Fluorescein fixation was observed on the corneas treated with the test substance.
- Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the obtained mean In Vitro Irritancy Score (IVIS) was 3, with individual IVIS of 5, 1 and 4 for each cornea. The mean IVIS indicates the test substance is not irritant to the eye. However, as two out of three corneas gave a prediction different from the mean of all three, results were considered borderline and no conclusion can be made.
Executive summary:

A study was conducted to determine the potential irritant and corrosive properties of the test substance to the eye according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at 32°C. A single experiment was performed using three corneas for each treated series (test substance, positive control and vehicle control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test substance was applied at the concentration of 10% (w/v) in the vehicle (0.9% NaCl), in a single experiment using a treatment time of 10 minutes and using the closed chamber treatment method. Vehicle and positive controls were treated concurrently. At the completion of the treatment period the epithelia were rinsed and the corneas were then incubated for 2 hours (± 10 minutes) at +32 °C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at 32 °C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities. Fluorescein fixation was observed on the corneas treated with the test substance. All acceptance criteria were fulfilled. The study was therefore considered as valid. Under the study conditions, the obtained mean In Vitro Irritancy Score (IVIS) was 3, with individual IVIS of 5, 1 and 4 for each cornea. The mean IVIS indicates the test substance is not irritant to the eye. However, as two out of three corneas gave a prediction different from the mean of all three, results were considered borderline and no conclusion can be made (Gerbeix, 2018).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion

A study was conducted to determine the skin corrosion potential of the test substance using reconstructed human epidermis model EPISKINTM(SM) according to OECD Guideline 431 and EU-Method B.40-BIS, in compliance with GLP. Disks of EPISKINTM(SM) (two units) were treated with test substance and incubated for 4 hours at room temperature. Exposure of test substance 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 substance. 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 substance is considered to be corrosive to skin. The experiment met the validity criteria, therefore the study was considered to be valid. Following exposure with the test substance the mean cell viability was 65.5 % compared to the negative control. This is above the threshold of 35%, therefore the test substance was considered as being non-corrosive. Under the study conditions, the results of the in vitro EPISKIN™(SM) model test with the test substance indicate that the test substance is non corrosive to the skin (Orovecz, 2017).

Skin irritation

A study was conducted to determine the skin irritation potential of the test substance using Reconstructed Human Epidermis (RHE) Test Method according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Three disks of EPISKINTM (SM were treated with the pre-warmed test substance and incubated for 15 minutes at room temperature. Exposure of the test substance 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 substance. The possible MTT interaction potential of the test substance was examined using two additional test substance 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. The experiment met the validity criteria, therefore the study was considered to be valid. Under the study conditions, following exposure with the test substance, the mean cell viability was 6.2 % compared to the negative control. This is below the threshold of 50 %, therefore the test substance was considered as being irritant to skin (Orovecz, 2017).

Eye irritation

A study was conducted to determine the potential irritant and corrosive properties of the test substance to the eye according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at 32°C. A single experiment was performed using three corneas for each treated series (test substance, positive control and vehicle control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test substance was applied at the concentration of 10% (w/v) in the vehicle (0.9% NaCl), in a single experiment using a treatment time of 10 minutes and using the closed chamber treatment method. Vehicle and positive controls were treated concurrently. At the completion of the treatment period the epithelia were rinsed and the corneas were then incubated for 2 hours (± 10 minutes) at +32 °C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at 32 °C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities. Fluorescein fixation was observed on the corneas treated with the test substance. All acceptance criteria were fulfilled. The study was therefore considered as valid. Under the study conditions, the obtained mean In Vitro Irritancy Score (IVIS) was 3, with individual IVIS of 5, 1 and 4 for each cornea. The mean IVIS indicates the test substance is not irritant to the eye. However, as two out of three corneas gave a prediction different from the mean of all three, results were considered borderline and no conclusion can be made (Gerbeix, 2018).

Justification for classification or non-classification

Skin irritation

Exposure to the test substance induced a mean cell viability of 6.2% compared to the negative control in an OECD Guideline 439 study. This is below the threshold of 50%, therefore the substance warrants classification as Skin Irrit. 2 – H315 (Causes skin irritation) according to CLP (EC 1272/2008) criteria.

Eye irritation

No negative effects were seen in an in vitro eye irritation study according to OECD Guideline 437 and was therefore not irritating under the conditions of this test. However, given its skin irritation properties and in line with classification for substances with similar structures, a conservative classification as Eye Irrit. 2 - H319 (Causes serious eye irritation) is proposed.