Registration Dossier

Administrative data

Description of key information

Weight of evidence: Skin irritating, precautionary and incorporating expert judgement, 2019

Skin corrosion, in vitro: non-corrosive: mean viability = 112.3% (3 minutes) and 98% (60 minutes), OECD TG 404, 2019

Skin irritation, in vitro: non-irritating, mean tissue viability 15-minute/42-hour incubation = 51.8%, OECD TG 439, 2019

Eye irritation, in vitro: non-eye corrosive/serious irritating, mean IVIS = 4.3 and mean permeability = 0.088, OECD TG 437, 2019

Eye irritation, in vitro: non-irritating, mean tissue viability = 72%, OECD TG 492, 2019

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08-08-2018 to 21-08-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017 ; signature: November 2017
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): Lot no.: 28646
- Production date: Not reported.
- Shipping date: Not reported.
- Delivery date: 21-08-2018
- Date of initiation of testing: ca. 22-08-2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT (prepared from a MatTek MTT-100 kit)
- Incubation time: The plate was incubated (37 °C, 5% CO2) for 3 hours.
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm (OD570)
- Filter: Not reported.
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 4.6% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Two (2), duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: The MTT solution containing the test item turned blue. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using freeze-killed tissues was performed. The results of the freeze-killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
- Procedure used to prepare the killed tissues (if applicable): Freeze-killed ; Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12-well plate and storing in a freezer (−14 to −30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, the MTT reducing test item was applied to two freeze-killed tissues per exposure period. In addition, two freeze-killed tissues per exposure period remained untreated. The untreated freeze-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.
- N. of replicates : Two (2), duplicate
- Method of calculation used: Interference by the test item relative to the corresponding negative control:
(a) 3 minutes exposure:
Mean of test item treated killed tissues (tkt) = 0.170 OD570
Mean of untreated killed tissues (ukt) = 0.227 OD570
The direct reduction by the test item relative to the negative control value:
(0.170 (tkt) – 0.227 (ukt)) / 1.796 (mean of negative control) = 0.0%*
*Negative value treated as 0.0%
(b) 60 minutes exposure:
Mean of test item treated killed tissues (tkt) = 0.257 OD570
Mean of untreated killed tissues (ukt) = 0.193 OD570
The direct reduction by the test item relative to the negative control value:
(0.257 (tkt) – 0.193 (ukt)) / 1.885 (mean of negative control) = 3.4%
The interference by the test item relative to the corresponding negative control was 0.0% after 3 minutes exposure and 3.4% after 60 minutes exposure. Therefore direct reduction was < 30% relative to the negative control and considered acceptable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if e.g. 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%.
- The test substance is considered to be non-corrosive to skin if e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Cut off points in accordance with OECD TG 431 and the GHS and CLP Classification systems.
- Skin corrosion is expressed as the remaining cell viability after exposure to the test substance at exposure times 3-minutes and 60-minutes, respectively. Where necessary, direct MTT reduction, colour interference and correction for background Isopropanol absorbance at OD570 (via measurement of triplicate blanks) was completed.

OTHER:
EpiDerm Skin Model (EPI-200, Lot no.: 28646). The test consists of topical application of the test item on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EpiDerm Skin Model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Preincubation:
Upon receipt of the Epiderm tissues, the sealed 24-well plate was stored in a refrigerator until use. The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of test item and rinsing:
- After pre-incubation of the EpiDerm tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure.
- The liquid test item was applied directly on top of the skin tissue. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 μL of sterile distilled water (negative control) was added to the first two tissues. 50 μL of the test item and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours.
- After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL of the test item
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl sterile distilled water (negative control)
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 8N KOH(aq) (pre-diluted)
Duration of treatment / exposure:
Observations are made 3-minutes and 1-hour post-test item application
Duration of post-treatment incubation (if applicable):
After rinsing: The plate was incubated (37 °C, 5% CO2) for 3 hour MTT incubation. Subsequently, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.
Number of replicates:
Two (n=2) for a 3-minute exposure to the test item and two for a 60-minute exposure
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minute exposure
Run / experiment:
mean (n=2)
Value:
112.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 3 minute exposure. Remarks: n=2 ; CV = 0.2% ; Score in terms of percentage of negative control.
Irritation / corrosion parameter:
% tissue viability
Remarks:
60 minute exposure
Run / experiment:
mean (n=2)
Value:
98
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 60 minute exposure. Remarks: n=2 ; CV = 7.2% ; Score in terms of percentage of negative control.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Corrected for by use of freeze-killed tissue replicates. The interference by the test item relative to the corresponding negative control was 0.0% after 3 minutes exposure and 3.4% after 60 minutes exposure. Therefore direct reduction was < 30% relative to the negative control and considered acceptable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Positive Control and Negative Control HCD data are presented within the full study report and the concurrent NC and PC was within the specified ranges.

Table 1. Mean absorption and tissue viability in the in vitro skin corrosion test with the test item

Tissue

Exposure Period

MeanOD570 of individual tissues (tvt)

Corrected Mean OD570 (tvt-[tkt-ukt])

Mean OD570 of duplicate tissues

Standard Deviation

Coefficient of Variation

(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.755

-

1.796

0.057

3.2

100*

1.836

-

60 Minutes

1.910

-

1.885

0.036

1.9

1.859

-

Positive Control

3 Minutes

0.092

-

0.081

0.016

na

4.5

0.069

-

60 Minutes

0.097

-

0.087

0.014

na

4.6

0.077

-

Test Item

3 Minutes

2.013

2.013

2.017

0.005

0.2

112.3

2.020

2.020

60 Minutes

1.818

1.754

1.848

0.132

7.2

98.0

2.005

1.941

OD = Optical density

tvt = Treated killed tissues

tkt = Untreated killed tissues

ukt =Untreated killed tissues

* = The mean % viability of the negative control tissue is set at 100%

na = Not applicable

 

3 minute exposure corrected mean OD570:

x̅ (0.163 + 0.176) = 0.170 (tkt) – x̅ (0.226 + 0.228) = 0.227 (ukt) = 0.000+
+Negative value treated as 0.0% to avoid artificially inflating the results.

60 minute exposure corrected mean OD570:

x̅ (0.280 + 0.233) = 0.257 (tkt) – x̅ (0.178 + 0.207) = 0.193 (ukt) = 0.064

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test item is not considered to be corrosive in the in vitro skin corrosion test using EpiDerm Skin Model.
Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS under GLP to assess the skin corrosion potential of the test item using a human three dimensional epidermal model EpiDerm (EPI-200). Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes together with a negative control and positive control. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). The mean OD570 for the negative control treated tissues was 1.796 for the 3 Minute exposure period and 1.885 for the 60 Minute exposure period. The relative mean tissue viability obtained after the 3-minute and 60-minute treatments with the test item (corrected for direct MTT reduction) compared to the negative control tissues was 112.3% and 98.0% respectively. The relative mean tissue viability for the positive control after the 3-minute and 60-minute treated tissues compared to the negative control tissues was 4.5% and 4.6%, respectively. The quality criteria required for acceptance of results in the test were satisfied. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 60-minute treatment the test item is not considered to be corrosive. Under the conditions of this study, the test item is not corrosive in the in vitro skin corrosion test.

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:
18-09-2018 to 11-02-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: August 2018; signature: November 2018
Species:
other: bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Recognised supplier.
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): 12 to 60 months old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Post-excision, placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics over ice packs.
- Time interval prior to initiating testing: < 24 hours. Corneas were prepared for testing immediately on same day arrival.
- indication of any existing defects or lesions in ocular tissue samples: None. Only corneas free from damage utilised.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL during transport.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
10 minutes at 32 ± 1ºC.
Duration of post- treatment incubation (in vitro):
A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
Number of animals or in vitro replicates:
Three (3) per test item, or negative or positive controls, respectively.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically. Additionally, only corneas with opacity < 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: 0.9% w/v Sodium chloride solution

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: Ethanol; > 99.8% purity

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: Yes. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

- POST-EXPOSURE INCUBATION: Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. Furthermore, following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The mean opacity and mean permeability values (OD492) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD492 value). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS
Irritation parameter:
in vitro irritation score
Run / experiment:
mean (n=3)
Value:
4.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No. The corneas treated with the test item were clear post treatment and slightly cloudy post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: 1. Ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean of for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 65.1. ACTUAL: PC IVIS = 45.4
2. sodium chloride 0.9% w/v solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values for bovine corneas treated with the respective negative control of the current historical control data (HCD). When testing liquids the negative control limit for opacity should be ≤2.30 and for permeability ≤0.41. ACTUAL: NC IVIS = 0.7, opacity ≤ 2.0 and permeability ≤ 0.008.

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

5

5

5

0

-

0.000

-

-

2

4

6

6

2

-

0.008

-

-

3

3

4

3

0

-

0.000

-

-

-

-

-

-

0.7 #1

-

0.003 #2

-

0.7

Positive Control

4

6

32

33

27

26.7

1.161

1.158

-

5

6

37

35

29

28.3

1.262

1.259

-

6

3

34

30

27

26.3

1.271

1.268

-

-

-

-

-

-

27.0 #3

-

1.229 #3

45.4

Test Item

7

4

7

8

4

3.3

0.025

0.022

-

8

4

9

5

5

4.3

0.117

0.114

-

10

4

7

6

2

1.3

0.129

0.126

-

-

-

-

-

-

3.0 #3

-

0.088 #3

4.3

OD = Optical Density

#1 = Mean of post-incubation – pre-treatment values

#2 = Mean permeability

#3 = Mean corrected value

Interpretation of results:
other: Inconclusive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test item no prediction can be made using Bovine Corneal Opacity and Permeability model. In vitro irritancy score (IVIS) was 4.3 (> 3.0 and < 55 in the prediction model).
Executive summary:

The study was performed according to OECD TG 437 and EU Method B.47 to assess the eye irritancy potential in accordance with GLP of the test item in isolated bovine corneas. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The ocular irritancy of the test substance was tested through topical application for 10 ± 1 minutes and post-exposure incubation for 120 minutes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol), was 45.4 and was within the historical positive control data range (29.6 to 65.1). The test item resulted in a mean in vitro irritancy score (IVIS) of 4.3 after 10 minutes of treatment. Since the IVIS was > 3.0 and < 55, no direct prediction could be made for the test item.

Applicant assessment indicates: under the conditions of this study the test item is not considered to be an eye corrosive in the Bovine Corneal Opacity and Permeability test due to no IVIS > 55 within the BCOP assay.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-02-2019 to 20-02-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
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:
MatTek Corporation EpiOcular™ Eye Irritation Test (OCL-200-EIT)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2015 ; signature: September 2015
Species:
other: human-derived epidermal keratinocyte tissues
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The test method is indicated as a validated test method recommended by OECD and/or recognised internationally for the sequential testing strategy for the assessment of ocular irritation.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27092, Certificate of Analysis provided in the full study report).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL of the test item
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.
Duration of treatment / exposure:
30 minutes at 37.0 ± 1.5°C
Duration of post- treatment incubation (in vitro):
overnight (ca. 17 hours) at 37 ± 1.5 °C
Number of animals or in vitro replicates:
Two tissues were used for each treatment group (test item, negative control and positive control).
Details on study design:
- Details of the test procedure used: See below.
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27092, Certificate of Analysis provided in the full study report).
- Doses of test chemical and control substances used: 50 µL test item / 50 µL deionised water (negative control) / 50 µL methyl acetate (positive control)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) for 30 minutes. After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular tissues. The tissues were incubated at standard culture conditions for 30 minutes. At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with PBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of PBS were used per test item. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue. At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
- Justification for the use of a different negative control than ultrapure H2O (if applicable): Not applicable.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Not applicable.
- Description of any modifications to the test procedure: Not applicable.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): The test item did not directly-reduce MTT or was colour inference in pre-checks conducted prior to the exposure.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Duplicate.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm
- Description of the method used to quantify MTT formazan: Versamax® Molecular Devices, 85737 Ismaning, Germany – spectrophotometrically.
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: According to the OECD TG 492 and GHS system.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The absolute mean OD570 of the positive control tissues was marginally outside the laboratory historical control data range: 6.86 – 48.38%. However, demonstrated a clear positive result (6.63%) which verified the sensitivity and functioning of the test system. The current HCD was provided in the full study report.
- Complete supporting information for the specific RhCE tissue construct used: Attached to the full study report.
- Reference to historical data of the RhCE tissue construct: Full historic control data is attached to the full study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The test assay is validated at the test laboratory and certification of technical proficiency from the model supplier is attached to the full study report.
- Positive and negative control means and acceptance ranges based on historical data: See above.
- Acceptable variability between tissue replicates for positive and negative controls: Yes, < 6.2 % for positive control, negative control and test item replicates.
- Acceptable variability between tissue replicates for the test chemical: Yes.
Irritation parameter:
other: Mean tissue viability (% of negative control)
Remarks:
n=2
Run / experiment:
mean - test item
Value:
72
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: Mean tissue viability (% of negative control)
Remarks:
n=2
Run / experiment:
mean - positive control
Value:
6.63
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test assay is validated at the test laboratory and certification of technical proficiency from the model supplier is attached to the full study report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: See 'details on study design' for further information.

Table 1.0 : Mean absorption and Mean Tissue Viability in the EPIOCULAR Test

Test Group

Tissue No.

Well 1 [OD570]

Well 2 [OD570]

Mean [OD570] (well 1 and

well 2)

Mean [OD570] blank corr. (well 1 and

well 2)

Mean [OD570] of T1 and T2

Tissue viabil. [%]

rel. viabil. of T1 and T2

Diff. of viabil. between T1 and T2 [p.p.]

Blank

 

0.035

0.036

0.035

 

Negative

Control

1

2.397

2.317

2.357

2.321

2.269

100.0

102.3

4.63

2

2.255

2.248

2.252

2.216

97.7

Positive

Control

1

0.172

0.169

0.171

0.135

0.150

6.63

6.0

1.33

2

0.203

0.198

0.201

0.166

7.3

Test Item

1

1.760

1.719

1.739

1.704

1.634

72.00

75.1

6.20

2

1.633

1.564

1.599

1.563

68.9

Acceptability of the Assay

a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.

b) The mean relative tissue viability of the positive control should be < 50% relative to the negative control.

c) The difference between the % tissue viabilities of the two identically treated replicates should be < 20%.

All assay acceptability criteria were met.

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is not considered to be an eye irritant.
Executive summary:

The study was performed to assess the eye irritancy potential of the test item using the EpiOcular EIT model under OECD TG 492 guideline in accordance with GLP. The test item was found to not directly reduce MTT or colour interfere in pre-test checks. The test item was topically applied for 30 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. The tissues were transferred to fresh medium and incubated for 120 minutes under standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The relative mean tissue viability obtained after 30 minutes treatment with the test item compared to the negative control tissues was 72%. The positive control had a mean cell viability of 6.63% after 30 minutes treatment. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptability criteria. The difference between the percentages of viability of two tissues treated identically was less than 20%, indicating that the test system functioned properly. Under the conditions of this study, since the test item relative mean viability was > 60% the test item is not predicted to be eye irritant.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Corrosion:

Key study : in vitro, OECD TG 431, 2019 : The study was performed to OECD TG 431 and EU Method B.40 BIS under GLP to assess the skin corrosion potential of the test item using a human three dimensional epidermal model EpiDerm (EPI-200). Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes together with a negative control and positive control. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). The mean OD570 for the negative control treated tissues was 1.796 for the 3 Minute exposure period and 1.885 for the 60 Minute exposure period. The relative mean tissue viability obtained after the 3-minute and 60-minute treatments with the test item (corrected for direct MTT reduction) compared to the negative control tissues was 112.3% and 98.0% respectively. The relative mean tissue viability for the positive control after the 3-minute and 60-minute treated tissues compared to the negative control tissues was 4.5% and 4.6%, respectively. The quality criteria required for acceptance of results in the test were satisfied. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 60-minute treatment the test item is not considered to be corrosive. Under the conditions of this study, the test item is not corrosive in the in vitro skin corrosion test.

Skin Irritation:

Key study : in vitro, OECD TG 439, 2019 : The study was performed to OECD TG 439 and EU Method B.46 under GLP to assess the skin irritation potential of the test item using a human three dimensional epidermal model (EPISKIN Small Model). Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 51.8% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 16.9%. The mean OD570 for the negative control treated tissues was 0.657 and the standard deviation value of the viability was 7.4%. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the viability was 1.4%. The positive control acceptance criteria were therefore satisfied. All acceptance criteria were considered to be met. Under the conditions of this study, the test item is not considered to be irritating to the skin. However, it was noted that this result was only just above the ≤ 50% threshold for triggering an irritant result and therefore it should be noted that even though this study was negative the test item has shown the potential to cause a degree of response to the skin.

 

Eye Irritation:

Key study : in vitro, OECD TG 437, 2019 : The study was performed according to OECD TG 437 and EU Method B.47 to assess the eye irritancy potential in accordance with GLP of the test item in isolated bovine corneas. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The ocular irritancy of the test substance was tested through topical application for 10 ± 1 minutes and post-exposure incubation for 120 minutes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol), was 45.4 and was within the historical positive control data range (29.6 to 65.1). The test item resulted in a mean in vitro irritancy score (IVIS) of 4.3 after 10 minutes of treatment. Since the IVIS was > 3.0 and < 55, no direct prediction could be made for the test item.

Applicant assessment indicates: under the conditions of this study the test item is not considered to be an eye corrosive in the Bovine Corneal Opacity and Permeability test due to no IVIS > 55 within the BCOP assay.

 

Key study : In vitro, OECD TG 492, 2019 : The study was performed to assess the eye irritancy potential of the test item using the EpiOcular EIT model under OECD TG 492 guideline in accordance with GLP. The test item was found to not directly reduce MTT or colour interfere in pre-test checks. The test item was topically applied for 30 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. The tissues were transferred to fresh medium and incubated for 120 minutes under standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The relative mean tissue viability obtained after 30 minutes treatment with the test item compared to the negative control tissues was 72%. The positive control had a mean cell viability of 6.63% after 30 minutes treatment. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptability criteria. The difference between the percentages of viability of two tissues treated identically was less than 20%, indicating that the test system functioned properly. Under the conditions of this study, since the test item relative mean viability was > 60% the test item is not predicted to be eye irritant.

Respiratory Irritation:

No data available.

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin irritation: category 2: H315

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.

 

For skin irritation, the substance demonstrated a potential for degree of skin irritation response within an available skin irritation in vitro assay (OECD TG 439, 2019) with a relative mean MTT viability of 51.8%. A precautionary classification has been applied on this basis.

 

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient mild irritating effects to the eye but which are insufficient for classification based on two available in vitro assays (BCOP, OECD TG 437, 2019 and EPIOCULAR, OECD TG 492, 2019).

 

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017)