Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The substance was determined to be non-corrosive to skin in an in vitro study performed according to OECD TG 431 using the EPISKIN™(SM) model.

The substance was determined to be non-irritant to skin in an in vitro study performed according to OECD TG 439 using the EPISKIN™(SM) model.

The substance was determined to be non-irritant to eyes in an in vitro study using isolated chicken’s eyes performed according to OECD TG 438.

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:
6 July - 7 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 440/2008, Annex Part B, B.40.bis (In Vitro Skin Corrosion: Human Skin Model Test)
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: epidermis
Justification for test system used:
The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial 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:
Human Skin
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-027, Expiry Date: 11 July 2016) 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.

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) and are documented in Appendix 2.

Kit Contents
Units: EPISKINTM(SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKINTM(SM) biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” (Batch No.: 16 MAIN3 044; Exp. Date: 13 July 2016); A flask of sterile “Assay Medium” (Batch No.: 16 ESSC 027; Exp. Date: 13 July 2016)

Kit Reception
In each case, the pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM(SM) kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

MTT solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 05 July 2016) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of
0.3 mg/mL (MTT working solution) immediately before use.

Acidified isopropanol
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Chemical action by the test material on MTT may mimic that of cellular metabolism 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
20 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours and then any colour change was observed:
-Test items which do not interact with MTT: yellow
-Test items interacting with MTT: blue or purple
After three hours incubation, yellow colour in the mixture was detected; therefore additional controls were not used in the experiment.

Check-method to detect the colouring potential of test item
Prior to treatment, the test item was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test item had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues. Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step:
MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

PERFORMANCE OF THE STUDY

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.
- 20 mg of test item was applied evenly to the epidermal surface of each of two test 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.

Chemicals might spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis). The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (23.4-24.8°C) covered with the plate lids.
Note: The negative and positive controls were also part of a concurrent study (CiToxLAB study code: 16/172-051BE) performed in the same experimental period using the same batch of chemicals and
same batch of skin units

Rinsing (Day 0)
After the incubation times, 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 for 3 hours, 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: 240 72800, Serial Number: 0920-14, Date of calibration: 02 September 2014, calibration is valid until September 2016) at the required wavelength on each day before use.

CALCULATIONS OF VIABILITY PERCENTAGES - see attachment

VALIDITY OF THE TEST
The mean OD value of the two negative control tissues should be ≥ 0.6 and ≤ 1.5 and negative control OD values should not be below historically established boundaries.
The acceptable mean viability % range for positive control is ≤ 20%.
The difference of viability between the two tissue replicates should not exceed 30%.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

INTERPRETATION OF TEST RESULTS
The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2015).
The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).
For 2 disks:
If both disks have mean viability of ≥35% = Non Corrosive
If both disks have mean viability of <35% = Corrosive (at the corresponding incubation period)
Otherwise:
If the mean value is ≥35% and the variability is less than 50% = Non Corrosive
If the mean value is <35% and the variability is less than 50% = Corrosive
Otherwise:
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
Duration of treatment / exposure:
4 hours
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
In this assay, two replicates per test item were used. Two negative controls and two positive controls were also run in each assay. Furthermore, as the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
92.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
87.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
90.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The mean OD value of the two negative control tissues was in the recommended range (0.975).
- Acceptance criteria met for positive control: yes. The two positive control treated tissues showed 0.5% viability demonstrating the proper performance of the assay.
- Acceptance criteria met for variability between replicate measurements: yes. The difference of viability between the two test item-treated tissue samples in the MTT assay was 4.9%.
- Range of historical values if different from the ones specified in the test guideline: Appendix 3
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of this in vitro EPISKIN model test, the test item is non-corrosive to skin.
Executive summary:

An in vitro skin corrosivity test of the 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 (detailed in 3.6. section). The corrosivity of the test item was evaluated according to OECD TG 431.

Disks of EPISKINTM(SM) (two units) were treated with the 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 the test item, the mean cell viability was 90.2% 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 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:
24 August - 26 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
6 July 2012
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Epidermis
Justification for test system used:
The EPISKIN (SM) model was considered suitable for the study because it has been validated for irritation testing in an international validation study and its use is recommended by the relevant guideline
for irritation testing (OECD 439).
Vehicle:
unchanged (no vehicle)
Details on test system:
Human Skin
EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.:16-EKIN-034, Expiry Date: 29 August 2016) 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) [7]. 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.

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) and are documented in Appendix 2.

Kit Contents
Units: EPISKINTM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKINTM (SM) biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” (Batch No.: 16 MAIN3 056; Exp. Date: 31 August 2016)
A flask of sterile “Assay Medium” (Batch No.: 16 ESSC 037; Exp. Date: 31 August 2016)

Kit Reception
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM (SM) kit was kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

MTT solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 23 August 2016) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of
0.3 mg/mL (MTT working solution) immediately before use.

Acidified Isopropanol
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

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
Approximately 10 mg of 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 (simulating a tissue humid environment). As the test items had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test items to stain the epidermis by using additional control tissues.

PERFORMANCE OF THE STUDY
Pre-incubation, application and rinsing and MTT test were performed under aseptic conditions (in sterile hood using sterile equipments).

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 20 mg of the 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: 16/237-043B), 16/264-043B and 16/265-043B 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 (± 0.5 min) at room temperature (25.5-26.7°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 (except of the two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere, protected from light.

Formazan extraction (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 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.

CALCULATIONS OF VIABILITY PERCENTAGES - see attachment

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18.
The SD calculated from individual % tissue viability values of the three test item treated replicates should be <18.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

INTERPRETATION OF TEST RESULTS
The irritation potential of test items can be classified according to the United Nations globally Harmonized System of Classification and Labelling of Chemicals [10], and a similar system is used in CLP [13]. In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- concentration: 5% w/v
Duration of treatment / exposure:
15 mins
Duration of post-treatment incubation (if applicable):
42 hours
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. Furthermore, as the test items were coloured, two additional test item-treated tissues were used for the non-specific OD (optical density or absorbance) evaluation.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
95.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
86.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
78.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
86.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues was in the recommended range (0.718). Standard deviation of the viability results for negative control samples was 2.8.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 5.6% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.8.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 8.5.
- Range of historical values if different from the ones specified in the test guideline: See Appendix 3
Interpretation of results:
GHS criteria not met
Conclusions:
in this in vitro EPISKIN model test the results indicate that the test item is non-irritant to skin.
Executive summary:

An in vitro skin irritation test of the 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 TG 439.

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2 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). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item.

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 the test item, the mean cell viability was 86.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test, the results indicate that the test item is non-irritant to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
15 August - 29 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
26 July 2013
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
08 December 2010
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
other: COBB 500 and ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS Kft., 9600 Sárvár, Rábasömjéni út. 129., Hungary
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): approximately 7 weeks old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Chicken heads were collected after slaughter in a commercial abattoir. 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).
- Time interval prior to initiating testing:<= 2 hours and 15 minutes of collection.
- indication of any existing defects or lesions in ocular tissue samples: No, all eyes were examined prior to testing to ensure they were in good condition.
- Indication of any antibiotics used: No data.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
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.
Number of animals or in vitro replicates:
3 per test
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
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 fluorescein-treated 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.

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.

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.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There 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.

EQUILIBRATION AND BASELINE RECORDINGS

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. In each experiment no changes in corneal thickness were observed. 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.

NUMBER OF REPLICATES: 3 for test item; 1 for negative control, 3 for positive control

NEGATIVE CONTROL USED: Physiological saline (Salsol solution, 0.9% (w/v) NaCl)

SOLVENT CONTROL USED (if applicable): Not applicable

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 30 mg for test item; 30 μL for negative control; 30 mg for positive control. Exposure time = 10 secs.

OBSERVATION PERIOD
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.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but
attempting to remove all residual the test item if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed. The test item treated eyes rinsed additional gentle rinsing with 20 mL saline after treatment.
- Indicate any deviation from test procedure in the Guideline: None

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and corneal opacity were measured at all time points. Haag-Streit BP 900 slit-lamp microscope was used for the measurements.
- Damage to epithelium based on fluorescein retention: 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.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting:
- Macroscopic morphological damage to the surface: 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.

SCORING SYSTEM:
- Mean corneal swelling (%): See attachment 'Evaluation'
- Mean maximum opacity score: See attachment 'Evaluation'
- Mean fluorescein retention score at 30 minutes post-treatment: See attachment 'Evaluation'

DECISION CRITERIA: The decision criteria as indicated in the TG were used.
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 1: mean maximum at 75 min.
Value:
1.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 1: mean maximum at 240 min
Value:
2.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment 1: mean maximum
Value:
0.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment 1: mean
Value:
0.83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 2: mean maximum at 75 min.
Value:
1.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 2: mean maximum at 240 min
Value:
1.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment 2: mean maximum
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment 2: mean
Value:
0.83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None noted
- Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control results were within the historical data range in each experiment (attached)
- Acceptance criteria met for positive control: The positive control results were within the historical data range in each experiment (attached)
- Range of historical values if different from the ones specified in the test guideline: (attached)
Interpretation of results:
GHS criteria not met
Conclusions:
Based on these in vitro eye irritation assays in isolated chicken eyes, the test item was non-irritant, UN GHS Classification: No Category.
Executive 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 TG 438 (26 July 2013). After the zero reference measurements, the eye was held in horizontal position and powdered 30 mg 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 each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

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 experiment. Thus, the experiments were considered to be valid. As the test item was solid, the observed negative result of the first experiment was confirmed by a second experiment.

Experiment I: No significant corneal swelling (≤ 5%) was observed during the four hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on two eyes. Slight fluorescein retention change (severity 0.5 or 1) was noted on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (≤ 5%) was observed during the four hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on three eyes. Slight fluorescein retention change (severity 0.5 or 1) was noted on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on these in vitro eye irritation assays in isolated chicken eyes, 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 irritation/corrosion

The potential of the substance to cause skin corrosion was evaluated according to OECD TG 431 using the EPISKINTM(SM) reconstructed human epidermis model.

Disks of EPISKINTM(SM) (two units) were treated with the 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 the test item, the mean cell viability was 90.2% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

The potential of the substance to cause skin irritation was evaluated according to OECD TG 439 using the EPISKINTM(SM) reconstructed human epidermis model.

Disks of EPISKINTM(SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2 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). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. 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 the test item, the mean cell viability was 86.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.

Eye irritation

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 TG 438. After the zero reference measurements, the eye was held in horizontal position and powdered 30 mg 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. In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

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 experiment. Thus, the experiments were considered to be valid. As the test item was solid, the observed negative result of the first experiment was confirmed by a second experiment.

Experiment I: No significant corneal swelling (≤ 5%) was observed during the four-hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on two eyes. Slight fluorescein retention change (severity 0.5 or 1) was noted on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (≤ 5%) was observed during the four-hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on three eyes. Slight fluorescein retention change (severity 0.5 or 1) was noted on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on these in vitro eye irritation assays in isolated chicken eyes, the test item was non-irritant, UN GHS Classification: No Category.

Justification for classification or non-classification

The substance was found to be non-corrosive to skin in an in vitro study performed according to OECD TG 431 and non-irritating to skin in an in vitro study performed according to OECD TG 439. It was non-irritating in an in vitro study using isolated chicken's eyes performed according to OECD TG 438.

On the basis of these studies, classification for skin or eye irritation is not required under CLP.