Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion

(OECD 431): non-corrosive

(OECD 439): irritating potential

Overall concusion: irritating

Eye irritation

(OECD 438): non-corrosive

(OECD 492): not irritating

Overall conclusion: not irritating

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:
05 - 11 May 2017
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:
adopted: 29 Jul 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ (EPI-200)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE)
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 25811
- Delivery date: 10 May 2017
- Date of initiation of testing: 10 May 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 ± 0.5 min exposure), 37 ± 1.5 °C (60 ± 5 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS (20 times) in order to remove any residual test material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, SoftMax Pro Enterprise v.4.7.1)
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 2.228 ± 0.215 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.16 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce the MTT solution, an additional functional check was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is greater or equal than 15%.
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N
Duration of treatment / exposure:
3 ± 0.5 min and 60 ± 5 min
Number of replicates:
duplicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
3 min exposure
Value:
108.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
60 min exposure
Value:
119.4
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
OTHER EFFECTS
- Direct-MTT reduction: Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water and thus passed the colour interference pre-test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.412 and 1.455).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour was <15% compared to the negative control (4.3%).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (values between 0.4% and 9.8%).

Table 2. Results after treatment with the test substance and controls

 

 

Exposure

interval (min)

Aborbance

at 570 nm *

Mean absorbance of 2 tissues

SD

Absorbance (% of negative control) **

CV

(%)

Rel. absorbance (% of negative control) **

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Negative

control

 

 

 

3

1.399

1.455

1.409

0.014

99.3

100.7

1.0

100.0

Positive

control

0.322

0.280

0.301

0.003

22.8

19.9

9.8

21.4

Test

substance

1.536

1.528

1.532

0.005

109.0

109.5

0.4

108.7

Negative

control

 

 

 

60

1.417

1.377

1.397

0.028

101.4

98.6

2.0

100.0

Positive

control

0.066

0.054

0.060

0.008

4.7

3.9

13.4

4.3

Test

substance

1.661

1.674

1.667

0.010

118.9

119.9

0.6

119.4

* Mean of three replicate wells after blank correction (blank: 0.035-0.037)

 ** Relative absorbance (rounded values): 100 × (mean absorbance test substance/positive control) / (mean absorbance negative control)

Interpretation of results:
other: non-corrosive according to OECD 431
Conclusions:
Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiDerm™ model, but no prediction on the skin irritation potential can be made and additional testing should be conducted for classification and labeling purposes.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Sep - 27 Oct 2017
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:
adopted: 28 Jul 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commision Regulation (EC) No 440/2008 of 30 May 2008; 1st ATP 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ (Epi-200)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (Epi-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 25850
- Delivery date: 24 Oct 2017
- Date of initiation of testing: 24 Oct 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; 25 min at room temperatur in a sterile bench
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.448 ± 0.05 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.16 h (acceptance criteria: 4.77 - 8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.


CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Since the test substance reduced MTT, it was applied to two freeze-killed tissues. In addition, two freeze killed tissues were left untreated (Note: The untreated killed controls show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue). The entire assay protocol is performed on the freeze-killed tissues in parallel to the assay performed with the live EpiDerm tissues. Data were then corrected as follows:
Data correction procedure
True viability = Viability of treated tissue – Interference from test chemical = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is ≤ 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% aqueous solution
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
approx. 41.5 h
Number of replicates:
triplicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
6.6
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The test item reduced MTT (test for direct MTT reduction), therefore an additional test with freeze-killed tissues had to be performed.
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.268, 1.323 and 1.250) was in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 5% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability values of the test substance, the positive and negative controls in the main test were < 6% (threshold of OECD 439: <18%), thus ensuring the validity of the study.

Table 1. Results after 1 h treatment

Treatment Group Tissue No. OD
570 nm
Well 1
OD
570 nm
Well 2
OD
570 nm
Well 3
Mean OD of 3 Wells Mean OD of 3 Wells blank corrected Mean OD of 3 tissues blank corrected Rel. Viability [%] Tissue 1, 2 + 3* Relative Standard Deviation [%] Mean Rel. Viability [%]**
Blank   0.038 0.039 0.037 0.038  
Negative Control 1 1.24 1.333 1.344 1.306 1.268   99.3    
2 1.357 1.369 1.359 1.361 1.323   103.6    
3 1.278 1.273 1.283 1.278 1.24 1.277 97.1 3.3 100
Positive Control 1 0.109 0.109 0.1 0.106 0.068   5.3    
2 0.11 0.1 0.098 0.103 0.065   5.1    
3 0.1 0.099 0.096 0.098 0.06 0.064 4.7 5.9 5
Test Item 1 0.115 0.108 0.111 0.111 0.074   5.8    
2 0.126 0.095 0.105 0.109 0.071   5.5    
3 0.11 0.112 0.11 0.111 0.073 0.072 5.7 1.9 6.6***
Negative Control Freeze killed Tissues 1 0.115 0.121 0.125 0.12 0.083   6.5    
2 0.122 0.124 0.126 0.124 0.087 0.085 6.8 2.9 6.7
Test Item Freeze killed Tissues 1 0.104 0.106 0.104 0.105 0.068   5.3    
2 0.116 0.117 0.118 0.117 0.08 0.075 6.3 11.8 5.8

* Relative viability (rounded values): 100 × (absorbance test item/positive control/negative control) / (mean absorbance negative control)

** Mean relative viability (rounded values): 100 × (absorbance test item/positive control/negative control) / (mean absorbance negative control)

*** corrected values

Interpretation of results:
other: irritating potential according to OECD 439
Conclusions:
Under the conditions of the Reconstructed Human Epidermis test the test substance is considered to possess an skin irritating potential, but no prediction on the skin corrosion potential can be made and additional testing should be conducted for classification and labeling purposes.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 Jun 2017
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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted 26 Jul 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Medicines & Healthcare products Regulatory Agency, UK
Test material information:
Composition 1
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Baileys Turkeys Ltd., Cheshire, UK
- Characteristics of donor animals: Chickens were approx. 56 days old and weighed 3 kg prior to slaughter.
- Storage, temperature and transport conditions of ocular tissue: Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Time interval prior to initiating testing: Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline.
- Indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
10 sec
Duration of post- treatment incubation (in vitro):
240 min
Reading time points: 30, 75, 120, 180 and 240 min (± 5 min)

Number of animals or in vitro replicates:
3 eyes for test group and positive control; 2 eyes for negative control
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
The integrity of the cornea was measured with a microscope after instillation of sodium fluorescein (2%, w/v). Acceptable values for fluorescein retention and corneal opacity scores are ≤ 0.5. Eyes acceptable for testing were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors.
Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner. Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ± 1.5 °C.
Once all eyes were placed in the superfusion apparatus, the eyes were examined again for corneal damage with the microscope. Corneal thickness measurements are taken with a depth measuring device on a microscope at the center of each cornea.
Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.


EQUILIBRATION AND BASELINE RECORDINGS
After the approval process the eyes were incubated for 45 min for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The test item was then rinsed from the eye using 20 mL of isotonic saline.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Haag-Streit BQ 900 slit-lamp microscope
- Damage to epithelium based on fluorescein retention: Haag-Streit BQ 900 slit-lamp microscope
- Swelling: Corneal thickness was measured with a depth measuring device no. 1 on a slit-lamp microscope.
- Macroscopic morphological damage to the surface: Haag-Streit BQ 900 slit-lamp microscope

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. The calculation was expressed in the following formula:
(corneal thickness at time (t) - corneal thickness at time =0) / (corneal thickness at time =0) x 100
Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.

- Mean maximum opacity score:
Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.

Opacity scores:
0 = No opacity
0.5 = Very faint opacity
1 = Scattered or diffuse areas; details of the iris are clearly visible
2 = Easily discernible translucent area; details of the iris are slightly obscured
3 = Severe corneal opacity; no specific details of the iris are visible; size of the pupil barely discernible
4 = Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention scores for all test eyes are calculated at the 30 min time interval only. These measurements are used for the overall classification for each test item.

Fluorescein retention scores:
0 = No fluorescein retention
0.5 = Very minor single cell staining
1 = Single cell staining scattered throughout the treated area of the cornea
2 = Focal or confluent dense single cell staining
3 = Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:
ICE classification criteria as explained in OECD TG 438 were used.
Irritation parameter:
cornea opacity score
Run / experiment:
240 min
Value:
1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE Class II
Irritation parameter:
fluorescein retention score
Run / experiment:
30 min
Value:
0.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
120 min
Value:
15.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE Class II
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, No fluorescein retention was noted in both negative control treated eyes.
- Acceptance criteria met for positive control: Yes, Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes.

Table 2: Ocular reactions in observed eyes

 

Score

ICE class

Test item

 

 

Maximal mean score for corneal opacity

1.0

II

Mean score of fluorescein retention

0.5

I

Maximan mean corneal swelling

15.70%

II

Positive control

 

 

Maximal mean score for corneal opacity

4.0

IV

Mean score of fluorescein retention

3.0

IV

Maximan mean corneal swelling

47.09%

IV

Negative control

 

 

Maximal mean score for corneal opacity

0.3

I

Mean score of fluorescein retention

0.0

I

Maximan mean corneal swelling

0.68%

I

Interpretation of results:
other: non-corrosive according to OECD 438
Conclusions:
The irritation potential of the test substance was assessed in the ICE assay. After treatment with the neat test substance corneal opacity, fluorescein retention and corneal swelling were classed as category II, I and II, respectively. According to OECD 438 the substance can be considered as non-corrosive, but no prediction on the eye irritation potential can be made and additional testing should be conducted for classification and labeling purposes.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Aug - 21 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
28 Jul 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test material information:
Composition 1
Species:
human
Strain:
other: EpiOcular™
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
post-exposure immersion: 12 min
post-treatment incubation: 120 min
Number of animals or in vitro replicates:
duplicate tissues for each treatment and control group
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ (MatTek Corporation, Bratislava, Slovakia), Lot No.: 27005

- Tissue viability: The quality of the final product was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.419 ± 0.105 (acceptance criteria: 1.1 - 3.0).

- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 15.97 min (acceptance criteria: 12.2 - 37.5 min).

- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure (37 °C), 12 min post-exposure immersion (room temperature), 120 min post-exposure (37 °C)

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary. Since the test item proved to reduce MTT a functional check on two freeze-killed tissues was performed under same experimental conditions as reported for the main assay.

- Description of the method used to quantify MTT formazan: The absorbance at 570 nm of each well was measured with a plate reader (Versamax Molecular Devices, Ismaning, Germany). No reference wavelength measurement was used.

- Decision criteria/Prediction model: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

- Acceptance criteria: The results are acceptable if
1) the negative control OD is > 0.8 and < 2.5
2) the mean relative viability of the positive control is below 50% of the negative control viability
3) the difference of viability between the two relating tissues of a single test substance is < 20% in the same run (for positive and negative control tissues and tissues of test substances). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to theviability of the relating negative control.This applies also to the freeze-killed tissues (single items and negative control) which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: mean relative absorbance (%)
Run / experiment:
30 min
Value:
75.4
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The test item proved to be an MTT reducer in the MTT pre-test. Therefore, additional tests with freeze killed viable tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water or isopropanol. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.601 and 1.580) was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean relative absorbance of the positive control is below 50% of the negative control viability (36.3%).
- The difference of rel. absorbance between the two relating tissues of a single item is < 20% (values between 0.3% to 6.9%) in the same run (for positive and negative control tissues and tissues of single test items).

Table 2. Results after 30 min exposure

Dose Group Absorbance
Well 1
(Tissue 1/2)
Absorbance
Well 2
(Tissue 1/2)
Mean Absorbance (Tissue 1/2) Mean Absorbance  Tissue 1 and 2  Mean Absorbance of 2 Tissues* Rel. Absorbance [%]
Tissue 1 and 2**
Absolute Value of the Difference of the Rel. Absorbances [%]
Tissue 1 and 2
Mean Rel. Absorbance
[%]**
Blank 0.036 0.036 0.036          
Negative Control 1.628 1.644 1.636 1.601 1.59 100.6 1.3 100
1.617 1.614 1.616 1.58 99.4
Positive Control 0.585 0.635 0.61 0.574 0.577 36.1 0.3 36.3
0.618 0.613 0.615 0.58 36.4
Test Item 1.176 1.155 1.165 1.13 1.185 71 6.9 75.4***
1.281 1.269 1.275 1.24 78
Negative Control 0.098 0.104 0.101 0.065 0.068 4.1 0.4 4.3
Freeze killed Tissues 0.107 0.107 0.107 0.071 4.5
Test Item Freeze killed Tissues 0.084 0.089 0.087 0.051 0.054 3.2 0.4 3.4
0.094 0.092 0.093 0.057 3.6

* Relative viability (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

** Mean relative viability (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** corrected values

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance is not considered to possess an irritating potential towards human cornea in the EpiOcular™ model.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin

The skin corrosion potential of the test substance was investigated by an in vitro skin corrosion test using a human skin model according to OECD Guideline 431 and in compliance with GLP (2017). After treatment with the test substance the tissue viability did not decrease (3 min exposure: 108.7%; 1 h exposure:119.4%) compared to the negative control. Therefore, the test substance is not considered to be corrosive towards human-derived epidermal keratinocytes, but no prediction on the skin irritation potential can be made and additional testing was conducted for classification and labeling purposes.

The skin irritation potential of the test substance was assessed by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2018). After treatment with the test substance for 60 min the tissue viability decreased (6.6%) compared to the negative control (threshold for classification 50%). Therefore, the test substance is considered to possess an skin irritant potential.

In the skin irritation/corrosion tests using reconstructed human epidermis, differing results concerning MTT reduction were obtained. In the test according to OECD 439 unspecific MTT reduction due to the test substance was observed, while in the test according to OECD 431 no MTT reduction was observed.

However, the obtained values for direct MTT reduction are considered to be very low. Therefore, even if unspecific MTT reduction is assumed for OECD 431, this would have led to a small correction of viability only, not impacting the overall outcome of the OECD 431 study (non-corrosive).

Eye

The eye irritation potential of the test substance was determined in an isolated chicken eye test (ICE) according to OECD Guideline 438 and in compliance with GLP (2017). Three isolated chicken eyes per group were exposed to a single application of the undiluted test substance for 10 sec using an application volume of 30 μL. Additional control groups were treated with physiological saline (negative control) or benzalconium chloride (positive control). Treatment with the test substance caused a mean corneal opacity score of 1.0 (ICE class II), a mean fluorescein retention score of 0.5 (ICE class I) and a mean corneal thickness of 15.77% (ICE class II). In conclusion, the test substance was not corrosive to the eye, but no prediction on the eye irritation potential can be made and additional testing was conducted for classification and labeling purposes.

The eye irritation potential of the test substance was further determined by an in vitro eye irritation test using a human cornea model according to OECD Guideline 492 and in compliance with GLP (2018). After treatment with the test substance for 30 min the tissue viability decreased to 75.4% compared to the negative control (threshold for classification≤60%). Therefore, the test substance is not considered to possess an eye irritating potential.

Justification for classification or non-classification

The available data on skin irritation of the test substance meet the criteria for classification as Skin Irrit. 2 (H315) according to Regulation (EC) 1272/2008.

The available data on eye irritation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.