Registration Dossier

Administrative data

Description of key information

Skin Irritation

Under the conditions of this study the results indicate that the test material is non-irritant to skin.

Skin Corrosion

Under the conditions of this study the test material is non-corrosive to the skin.

Eye Irritation

Under the conditions of this study the test material is not irritating to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2016 to 18 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN™ (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ (SM)
- Manufacturer: SkinEthic, France
- Tissue batch number(s): 16-EKIN-046
- 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.
- Expiry date: 21 November 2016
- The EPISKIN™ (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.

CHECK FOR DIRECT MTT REDUCTION
Approximately 10 mg of test material 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 materials which do not react with MTT: Yellow,
Test materials reacting with MTT: Blue or purple.
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test material did not react with MTT and therefore the use of additional controls was not necessary.

CHECK FOR COLOURING POTENTIAL OF THE TEST MATERIAL
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol (simulating a tissue humid environment). As the test materials had an intrinsic colour, further evaluation to detect colouring potential was necessary. Non-specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non specific OD evaluation. This tissue followed the same test material 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 material that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

MAIN 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)
As the test material was solid, 10 μL of distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 10 mg of the test material was applied evenly to the epidermal surface. If necessary, the test material 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.
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).
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.9 to 26.0 °C).


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (23.9 to 26.0 °C)
- Temperature of post-treatment incubation: 37 °C

NUMBER OF REPLICATES FOR THE TEST: 3

REMOVAL OF TEST MATERIAL AND CONTROLS
After the 15 minutes incubation time, the EPISKIN™ (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 AND FORMAZAN EXTRACTION (DAY 2)
After the 42 hour incubation, all EPISKIN™ (SM) units (except 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). The transferred EPISKIN™ (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.
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. 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.
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.

CALCULATIONS OF VIABILITY PERCENTAGES

FOR NORMAL TEST MATERIALS:
-Blank: The mean of the six blank OD values was calculated
-Negative control: Individual negative control OD values (NCraw) were corrected with the mean blank OD: OD Negative Control (ODNC) = ODNCraw – ODblank mean. The mean corrected OD of the 3 negative control samples were calculated: This value corresponds to 100 % viability
-Positive control: Individual positive control OD values (PCraw) were corrected with the mean blank
OD: OD Positive Control (ODPC) = ODPCraw – ODblank mean. The mean corrected OD of the 3 positive control samples were calculated. The % viability for each positive control replicate was calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
The mean value of the 3 individual relative viability % for positive control was calculated:
Mean PC % = (%PC1 +%PC2 +%PC3) / 3

-Test material: Individual test material OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
The mean corrected OD of the 3 test material samples were calculated. The % viability for each test material replicate was calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
The mean value of the 3 individual relative viability % for test material was calculated: Mean TT % = (%TT1 +%TT2 +%TT3) / 3

FOR TEST MATERIALS WITH MTT-INTERACTING POTENTIAL:
Test materials that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability% as detailed below:
-Non-specific MTT reduction calculation (NSMTT%):NSMTT (%) = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: Negative control killed tissues OD
ODKT: Test material treated killed tissues OD
ODNC: Negative control OD
If NSMTT% is ≤ 30 %, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: Test material treated viable tissues
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3
If NSMTT% is > 30 % relative to the negative control, then additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

FOR TEST MATERIALS WITH COLOURING POTENTIAL
For test materials detected as able to stain the tissues, the non-specific OD due to the residual chemical colour (unrelated to mitochondrial activity) is evaluated and subtracted before calculation of the “true” viability % as detailed below:
Non-specific Colour % with viable tissues (NSCliving %):= (mean ODCTV / mean ODNC)×100
ODCTV: Test substance treated viable tissues (not incubated with MTT)
ODNC: Negative control OD (incubated with MTT)
If NSCliving % is ≤ 5 % then the normal calculation mode is used.
If NSC % is > 5 % and ≤ 30 %, then true MTT metabolic conversion (TODTT) is undertaken as follows.
TODTT = [ODTT – mean ODCTV]
ODTT: Test substance treated viable tissue (incubated with MTT)
ODCTV: Test substance treated viable tissue (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated: (% RV 1 +% RV 2 +% RV 3) / 3
If NSC living % is > 30 % relative to the negative control, additional steps must be undertaken if possible, or the test substance must be considered as incompatible with the test.

FOR TEST MATERIALS WITH BOTH MTT INTERACTING POTENTIAL AND COLOURING POTENTIAL
Test substances detected as able to stain the tissues and interfere with MTT also require a third set of controls before calculation of the “true” viability %.
Non-specific Colour % with killed tissues (NSCkilled%) = (mean ODCTK / mean ODNC)×100
ODCTK: Test substance treated killed tissues (not incubated with MTT)
ODNC: Negative control OD (incubated with MTT)
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: Test substance treated viable tissues (incubated with MTT)
ODKT: Test substance treated killed tissues OD
ODKNC: Negative control killed tissues OD
ODCTV: Test substance treated viable tissues (not incubated with MTT)
ODCTK: Test substance treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test substance is calculated = (% RV 1 + % RV 2 + % RV 3) / 3

INTERPRETATION OF TEST RESULTS
-The test material 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: 10 mg applied with 10 µL distilled water.

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 5 % w/v (prepared in distilled water)
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 ± 1 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
103.1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
ADDITIONAL CONTROLS
-As no colour change was observed after three hours of incubation of the test material in MTT working solution, the test material did not interact with MTT and additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
-As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density of tissues were 0.006, Non-specific Colour % was calculated as 0.8 %. This value was below 5 %, therefore additional data calculation was not necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The OD values for the test material treated skin samples showed 103.1 % relative viability.

Validity of the Test

-After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

-The mean OD value of the three negative control tissues was in the recommended range (0.744). Standard deviation of the viability results for negative control samples was 4.7.

-The positive control treated tissues showed 6.3 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.6.

-The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 2.6.

-The mean OD value of the blank samples (acidified isopropanol) was 0.046.

-All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Table 1. Optical Density (OD) and the calculated relative viability % of the samples

Substance

Optical density

Viability (% RV)

 

Measured

Blank corrected

Negative control: PBS

1

0.790

0.744

100.1

2

0.754

0.708

95.2

3

0.824

0.778

104.7

Mean

-

0.744

100.0

Positive control: 5 % (w/v) SDS solution

1

0.087

0.041

5.5

2

0.085

0.039

5.3

3

0.107

0.061

8.2

Mean

-

0.047

6.3

Test material

1

0.832

0.786

105.7

2

0.811

0.765

102.8

3

0.794

0.748

100.6

Mean

-

0.767

103.1

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.
Executive summary:

The potential of the test material to cause skin irritation was determined in accordance with the standardised guidelines OECD 439, EU Method B.46 and EPISkin SOP Version 1.8 under, GLP conditions using a human skin model.

EPISKIN (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. Disks of EPISKIN (SM) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material 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 test was performed in triplicate.

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 material. For each treated tissue, the viability was expressed as a % relative compared 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 material is considered to be irritant to skin.

Following exposure to the test material, the mean cell viability was 103.1 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, under the conditions of this study the results indicate that the test material is non-irritant to skin (No Category based on the skin corrosivity test), UN GHS Classification: Non-classified.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 October 2016 to 14 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN™(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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM)
- Tissue batch number(s): 16-EKIN-041
- Expiry date: 17 October 2016
- 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.
- The EPISKIN™(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.

CHECK FOR DIRECT MTT-REDUCTION
Approximately 20 mg of test material 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 materials which do not react with MTT: Yellow
Test materials reacting 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 FOR COLOURING POTENTIAL OF THE TEST MATERIAL
Prior to treatment, the test material was evaluated for their intrinsic colour or ability to become coloured in contact with water and/or isopropanol. As the test material 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 material to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non specific OD evaluation. These tissues followed the same
test material 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 material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

MAIN 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 material was applied evenly to the epidermal surface of each of two test units and then 100 μL physiological saline was added to the test material to ensure good contact with the epidermis.
50 μL of physiological saline was added to each of the two negative control skin units and 50 μL of glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (23.2 to 25.5 °C) covered with the plate lids.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (23.2 to 25.5 °C)
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the incubation time all test material 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 AND FORMAZAN EXTRACTION
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.
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.

CALCULATIONS OF VIABILITY PERCENTAGES
-Blank: The mean of the 6 blank OD values was calculated

Negative control:
–Individual negative control OD values (NCraw) were corrected with the mean blank OD: OD Negative Control (ODNC) = ODNCraw – ODblank mean
–The corrected mean OD of the 2 negative control values was also calculated: this corresponds to 100 % viability

Positive control:
–Individual positive control OD values (PCraw) were corrected with the mean blank OD: OD Positive Control (ODPC) = ODPCraw – ODblank mean
–The corrected mean OD of the 2 positive control values was calculated
–The % viability for each positive control replicate was calculated relative to the mean negative control: Positive Control1 % = (ODPC1 / mean ODNC) ×100, Positive Control2 % = (ODPC2 / mean ODNC) ×100
–The mean value of the 2 individual viability % for positive control was calculated: Mean PC % = (PC1 % + PC2 % ) / 2

Test material:
– Individual test material OD values (TTraw) were corrected with the mean blank OD: OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 2 test material values was calculated
– The % viability for each test material replicate was calculated relative to the mean negative control:
Treated Tissue1 % = (ODTT1 / mean ODNC) ×100
Treated Tissue2 % = (ODTT2 / mean ODNC) ×100
–The mean value of the 2 individual viability % for test material was calculated: Mean TT % = (TT1 % + TT2 %) / 2
–The variability for 2 disks is calculated as: (Disk1-Disk2)/((Disk1+Disk2)/2) x 100 %

CALCULATIONS FOR TEST MATERIALS WITH MTT-INTERFERING POTENTIAL
Test materials that interfere with MTT can produce non specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value was corrected by the result of the additional controls before calculation of viability% as follows:
Non specific MTT reduction calculation (NSMTT %): = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: Negative control treated killed tissues OD
ODKT: Test material treated killed tissues OD
ODNC: Negative control OD
If NSMTT % is ≤ 50 %, then true MTT metabolic conversion (TODTT) has to be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: Test material treated viable tissues – The % relative viability (RV%) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test material is calculated = (RV1 % + RV2 %) / 2
If NSMTT % is > 50 % relative to the negative control: additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

CALCULATIONS FOR TEST MATERIALS WITH COLOURING POTENTIAL
For test materials detected as able to stain the tissues the non specific OD was evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability % as detailed below: Non Specific Colour % (NSCliving %) = (mean ODCTV / mean ODNC)×100
ODCTV: Test material treated viable tissue (not incubated with MTT)
ODNC: Negative control OD (incubated with MTT)
If NSCliving % is ≤ 5 % then the normal calculation mode was used.
If NSC living % is > 5 % a nd ≤ 5 0 %, then additional correction (TODTT) had to be
undertaken as follows: TODTT = [ODTV - ODCTV]
ODTT: Test material treated viable tissue (incubated with MTT)
ODCTV: Test material treated viable tissue (not incubated with MTT)
– The % relative viability (RV% %) for each test material replicate was calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test material was calculated: Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSC living % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

CALCULATIONS FOR TEST MATERIALS WITH MTT INTERACTING AND COLOURING POTENTIAL
For test materials detected as able to both stain the tissues and interfere with MTT may also require a third set of controls before calculation of the “true”viability %.
Non Specific Colour % with killed tissues (NSCkilled %) = (mean ODCTK / mean ODNC)×100
ODCTK: Test material treated killed tissues (not incubated with MTT)
ODNC: Negative control OD (incubated with MTT)
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: Test material treated viable tissues (incubated with MTT)
ODKT: Test material treated killed tissues OD
ODKNC: Negative control killed tissues OD
ODCTV: Test material treated viable tissues (not incubated with MTT)
ODCTK: Test material treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2% = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test material is calculated = (RV1 % + RV2 %) / 2

INTERPRETATION OF TEST RESULTS
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: 20 mg (with 100 µL of physiological saline to ensure good contact).

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 0.9 % w/v

POSITIVE CONTROL
- Amount(s) applied: 50 µL
Duration of treatment / exposure:
4 hours ± 10 minutes
Duration of post-treatment incubation (if applicable):
3 hours with MTT
Number of replicates:
Two
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
94.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
ADDITIONAL CONTROLS
As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.007, Non Specific Colour % (NSCliving %) was calculated as 0.8 %. This is below the threshold of 5 %, therefore correction due to colouring potential was not necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The mean OD value for the test material treated skin samples showed a 94.5 % relative viability.

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 (0.780).

-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 material-treated tissue samples in the MTT assay was 2.4 %.

-The mean OD value of the blank samples (acidified isopropanol) was 0.047.

All these parameters were within acceptable limits and therefore the study was considered to be valid.

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Substance

Optical density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative control: Physiological saline

1

0.794

0.747

95.7

2

0.860

0.814

104.3

Mean

-

0.780

100.0

Positive control: Glacial acetic acid

1

0.055

0.008

1.0

2

0.051

0.005

0.6

Mean

-

0.006

0.8

Test material

1

0.793

0.746

95.6

2

0.775

0.728

93.3

Mean

-

0.737

94.5

Interpretation of results:
other: Not corrosive in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.
Executive summary:

The potential of the test material to cause skin corrosion was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.Bis, under GLP conditions using the human skin model.

EPISKIN(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.

Disks of EPISKIN(SM) were treated with the test material 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. The study was performed in duplicate.

Physiological saline (0.9 % (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively and were also performed in duplicate. Two additional disks were used to provide an estimate of colour contribution (NSCliving %) from the test material. 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 material is considered to be corrosive to skin.

Following exposure with the test material, the mean cell viability was 94.5 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, under the conditions of this study the test material is non corrosive to the 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:
10 October 2016 to 24 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.

Species:
chicken
Strain:
other: COBB 500 and ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption.
- Characteristics of donor animals: Approximately 7 weeks old
- Storage, temperature and transport conditions of ocular tissue: Heads were collected by a slaughter house technician and transfered to the testing laboratory at ambient temperatures at the earliest convenience. 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 the testing laboratory and processed within 2 hours of collection.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 mg
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
Observations were made at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse, minor variations within approximately ± 5 minutes were considered acceptable.
Number of animals or in vitro replicates:
One eye was treated with physiological saline, three eyes with the test material and another three eyes with powdered imidazole in each experiment.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED 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 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.
-Preparation: 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.
-Examination and acclimatisation: The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position. 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, acclimatisation 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 acclimatisation and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatisation 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. No changes in thickness (0.0 %) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
Negative control: One
Test material: Three
Positive control: Three.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 30 mg of test material / 30 µL negative control / 30 mg positive control, exposure time of 10 seconds.

METHOD
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 test material was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test material, taking care not to damage or touch the cornea.
In each experiment the negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole.

REMOVAL OF TEST SUBSTANCE
-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 material or positive control material remaining on the cornea was observed. The test material treated eyes were rinsed with additional gentle rinsing with 2 or 3 x 20 mL saline after treatment in each experiment.

OBSERVATION PERIOD: Observations were made at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse, minor variations within approximately ± 5 minutes were considered acceptable.

METHODS FOR MEASURED ENDPOINTS:
- 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 Bern 900 slit-lamp microscope was used for the measurements.
- At the end of the procedure, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10 % neutral buffered formalin) used for potential histopathology and stored at room temperature.

EVALUATION/ SCORING SYSTEM:
- Corneal swelling was calculated according to the following formulae:

CS at time t = [(CT at time t – CT at t=0) / CT at t=0 ] x100

Mean CS at time t = [FECS (at time t) + SECS (at time t) + TECS (at time t)] / 3

Where:
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point
Small negative numbers for swelling (0 to -5 %) following application are evaluated as class I. Large negative numbers (>12 % below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

- Cornea opacity was calculated according to the following formulae:

ΔCO at time t = CO at time t – CO at t=0

Mean ΔCOmax = [FECOmax(30min to 240min) + SECOmax(30min to 240min) + TECOmax(30min to 240min)] / 3

Where:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the posttreatment rinse
CO at t=0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

- Fluorescein retention was calculated according to the following formulae:

ΔFR at time t = FR at time t – FR at t=0

Mean ΔFR = [FEFR (30min) + SEFR(30min) + TEFR(30min)] / 3

Where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 1 Mean (at up to 75 min)
Value:
2.2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 1 Mean (at up to 240 min)
Value:
2.2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment 1 Mean
Value:
0.67
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment 1 Mean
Value:
0.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 2 Mean (at up to 75 min)
Value:
0.6
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 2 Mean (at up to 240 min)
Value:
1.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment 2 Mean
Value:
0
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment 2 Mean
Value:
0.33
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.
Other effects / acceptance of results:
VALIDITY OF THE TEST
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Positive control:
Experiment I
Mean maximum corneal swelling at up to 75 min: 12.0 %
Mean maximum corneal swelling at up to 240 min: 28.3 %
Mean maximum corneal opacity: 4.0
Mean fluorescein retention: 3.0
Other Observations: Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Experiment II
Mean maximum corneal swelling at up to 75 min: 10.2 %
Mean maximum corneal swelling at up to 240 min: 25.7 %
Mean maximum corneal opacity: 4.0
Mean fluorescein retention: 3.0
Other Observations: Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Negative control:
Experiment I
Mean maximum corneal swelling at up to 240 min: -1.6 %
All other parameters assessed: 0.0
Experiment II: 0.0 for all parameters assessed.

Summary of Results

Experiment 1

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

2.2 %

I

Mean maximum corneal swelling at up to 240 min

2.2 %

I

Mean maximum corneal opacity

0.67

II

Mean fluorescein retention

0.50

I

Other Observations

Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.

Overall ICE Class

2 x I 1 x II

Experiment 2

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.6 %

I

Mean maximum corneal swelling at up to 240 min

1.7 %

I

Mean maximum corneal opacity

0.0

I

Mean fluorescein retention

0.33

I

Other Observations

Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.

Overall ICE Class

3 x I

Interpretation of results:
other: Not classified according to EU criteria.
Conclusions:
Under the conditions of the study the test material is not irritating to the eye.
Executive summary:

The potential of the test material to cause eye irritation was determined in accordance with the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions using the isolated chicken eye.

After the zero reference measurements, the eye was held in horizontal position and 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 the study, three test material 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 experiment was considered to be valid.

Experiment I: No significant corneal swelling (mean ≤ 5 %) was observed during the four-hour observation period on test material treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on three eyes. Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 120 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (mean ≤ 5 %) was observed during the four hour observation period on test material treated eyes. No cornea opacity change was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on two eyes. Minimal amount of test material was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 120 minutes after the post-treatment rinse.

Under the conditions of the study the test material is not irritating to the eye.

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

Additional information

Skin Corrosion

The potential of the test material to cause skin corrosion was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.Bis, under GLP conditions using the human skin model. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

EPISKIN(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.

Disks of EPISKIN(SM) were treated with the test material 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. The study was performed in duplicate.

Physiological saline (0.9 % (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively and were also performed in duplicate. Two additional disks were used to provide an estimate of colour contribution (NSCliving %) from the test material. 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 material is considered to be corrosive to skin.

Following exposure with the test material, the mean cell viability was 94.5 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, under the conditions of this study the test material is non corrosive to the skin.

Skin Irritation

The potential of the test material to cause skin irritation was determined in accordance with the standardised guidelines OECD 439, EU Method B.46 and EPISkin SOP Version 1.8 under, GLP conditions using a human skin model. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

EPISKIN(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. Disks of EPISKIN(SM) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material 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 test was performed in triplicate.

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 material. For each treated tissue, the viability was expressed as a % relative compared 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 material is considered to be irritant to skin.

Following exposure to the test material, the mean cell viability was 103.1 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, under the conditions of this study the results indicate that the test material is non-irritant to skin (No Category based on the skin corrosivity test), UN GHS Classification: Non-classified.

Eye Irritation

The potential of the test material to cause eye irritation was determined in accordance with the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions using the isolated chicken eye. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

After the zero reference measurements, the eye was held in horizontal position and 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 the study, three test material 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 experiment was considered to be valid.

Experiment I: No significant corneal swelling (mean ≤ 5 %) was observed during the four-hour observation period on test material treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on three eyes. Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 120 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (mean ≤ 5 %) was observed during the four hour observation period on test material treated eyes. No cornea opacity change was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on two eyes. Minimal amount of test material was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 120 minutes after the post-treatment rinse.

Under the conditions of the study the test material is not irritating to the eye.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin corrosion or eye irritation.