Registration Dossier

Administrative data

Description of key information

Skin irritation: Non-irritant to skin; OECD 439; Spohr, C. (2018)

Eye corrosion: No prediction could be made with regards to eye corrosion; OECD 437; Spohr, C. (2018)

Eye irritation: Non-irritant to eye; OECD 492; Spohr, C. (2018)

The substance does not meet the criteria for classification under local toxicity in accordance with GHS and Regulation (EC) No 1272/2008 (CLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 August 2018 - 17 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
yes
Remarks:
deviations were considered to have not affected the integrity or validity of the study.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
yes
Remarks:
deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: applied as supplied
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: n/a
Test system:
human skin model
Source species:
human
Cell type:
other: Epider RHE model supplied by MatTek Slovakia
Cell source:
other: The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200- SIT Kit
- Tissue batch number(s): 28644
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: August 14, 2018
- Date of initiation of testing: August 1, 2018


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

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, version 4.7.1) with a 570 nm filter.
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤20% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%. The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.8 and ≤ 2.8 and the SD value of the percentage viability is ≤18%.
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: Not reported
- Reproducibility: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- n/a, test item did not reduce MTT.
- Procedure used to prepare the killed tissues (if applicable): n/a
- N. of replicates : n/a
- Method of calculation used: n/a

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is equal to or less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure is greater than or equal to 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): unchanged- applied as supplied.

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % w/v
Duration of treatment / exposure:
15 min test item exposure
Duration of post-treatment incubation (if applicable):
42 h followed by 3 h MTT incubation
Number of replicates:
3 per test group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 min exposure
Value:
97.78
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is ≥ 0.8 and ≤ 2.8, and the SD value of the percentage viability is ≤18%.
- Acceptance criteria met for positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤20% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
- Acceptance criteria met for variability between replicate measurements: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.
- Range of historical values if different from the ones specified in the test guideline: Laboratory historical control data was performed to verify the functioning of the test system

Table 1. Results after treatment with AD-464 and the controls

Treatment Group

Tissue No.

OD 570 nm

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*

Standard Deviation

Mean Rel. Viability [%]*

Blank

 

0.036

 

0.036

0.036

0.036

 

Negative Control

1

1.883

 

1.996

 

2.032

 

1.971

 

1.935

 

1.885

 

102.634

 

3.9

 

100.0

 

2

1.931

 

1.961

 

1.978

 

1.956

 

1.920

 

101.877

 

3

1.820

 

1.806

 

1.882

 

1.836

 

1.800

 

95.488

 

Positive Control

 

1

0.095

 

0.092

 

0.093

0.093

0.057

 

0.060

 

3.036

 

0.1

 

3.20

 

2

0.097

 

0.098

0.097

0.097

0.061

 

3.245

 

3

0.098

 

0.100

0.098

0.098

0.062

 

3.312

 

Test Item

 

1

1.769

 

1.842

 

1.805

1.805

1.769

 

1.843

 

93.868

 

4.6

 

97.78

 

2

1.965

 

1.991

 

1.971

 

1.976

 

1.940

 

102.901

 

3

1.868

 

1.867

 

1.834

 

1.856

 

1.820

 

96.565

 

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study the test item was classified as non-irritant (UN GHS and Regulation (EC) 1272/2008 classification criteria not met).
Executive summary:

OECD 439 (2018) - The skin irritation potential of the test item was assessed using a Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

 

Triplicate tissues were exposed to the test item for 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a microplate reader (Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1).

Each three tissues of the human skin model EpiDermTM were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item AD-464 the mean relative viability value decreased to 97.78% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, AD-464 is non-irritant to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
07 August 2018 - 30 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted to GLP and standard giudeline with all criteria 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:
2017
Deviations:
yes
Remarks:
Deviation is considered to not affect the outcome or the integrity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
Species:
human
Details on test animals or tissues and environmental conditions:
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (28th August 2018) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.

Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (about 16 - 24 hours).
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
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a
Duration of treatment / exposure:
30 minutes
Observation period (in vivo):
N/A
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Each group tested in duplicate
Details on study design:
- Details of the test procedure used

Pre Test Assessments

The test items ability to directly reduce MTT was assessed. For this purpose approximately 50 µl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. The result indicated that the test item did not have MTT reducing effects. An additional test with freeze-killed tissues did not have to be performed.

The photometric properties of the test item after contact with water and isopropanol were assesed. For this purpose each 50 µL of the test item were added to 1.0 ml of water and to 2 ml isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least one hour, the isopropanol mixture or for 2 to 3 hours at room temperature. The result indicated that the test item did not dye the water or isopropanol and would therefore not interfere with the photometric assessment of MTT during the main test.

Main Test

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, 95% RH) 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 Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).

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 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 glass tube containing 1 ml of warm Assay Medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

After post-treatment incubation of 120 minutes the MTT assay was performed.

At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.

The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

- RhCE tissue construct used, including batch number: EpiOcular kit (Lot: 23574). Supplied by MatTek Coorp., Slovakia.

- Doses of test chemical and control substances used: 50 µL applied topically.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Tissues exposed for 30 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH. Tissues were immersed for 2 seconds during the rinsing process. Post exposue incubation was 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

- Justification for the use of a different negative control than ultrapure H2O (if applicable): N/A

- Justification for the use of a different positive control than neat methyl acetate (if applicable): N/A

- Description of any modifications to the test procedure: N/A

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): N/A

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per test group (test item, positive and negative control). 1 blank tissue prepared with no treatment.

- 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: See above.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): N/A

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated. The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified. The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model. If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes

- Complete supporting information for the specific RhCE tissue construct used: Yes

- Reference to historical data of the RhCE tissue construct: Yes

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Yes

- Positive and negative control means and acceptance ranges based on historical data: Yes

- Acceptable variability between tissue replicates for positive and negative controls: Yes

- Acceptable variability between tissue replicates for the test chemical: Yes
Irritation parameter:
other: Spectrophotometric mean relative absorbance of MTT solution at 570 nm
Run / experiment:
Mean value
Value:
104.35
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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD is > 0.8 and < 2.5 (1.927)
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (46.58%)
- Range of historical values if different from the ones specified in the test guideline: n/a

Table 1: Assessment of Colour Interference with the MTT endpoint

Treatment Group

OD 570 nm Well 1

OD 570 nm Well 2

Mean OD of 2 Wells

Mean OD of 2 Wells blank corrected

 

Evaluation Mean OD570 (blank corrected)
> 0.08

Blank Aqua Deion.

0.037

0.0376

0.037

 

Test Item + Aqua Deion.

0.060

0.109

0.085

0.048

no

Blank Isopropanol

0.036

0.041

0.039

 

Test Item+ Isopropanol

0.037

0.037

0.037

-0.002

no

The mean OD was < 0.08 and therefore viable tissues were not necessary in the main experiment

Table 2: Assessment of Colour Interference with the MTT endpoint

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

 

 

0.036

 

0.035

 

0.036

 

 

Negative Control

 

1

2.460

 

2.528

 

2.494

 

2.458

 

2.366

 

100.0

 

103.9

 

7.78

 

2

2.316

 

2.304

 

2.310

 

2.274

 

96.1

 

Positive Control

1

1.027

 

1.106

 

1.066

 

1.031

 

1.102

 

46.58

 

43.6

 

6.05

 

2

1.195

 

1.224

 

1.209

 

1.174

 

49.6

 

Test Item

1

2.401

 

2.491

 

2.446

 

2.410

 

2.469

 

104.35

 

101.9

 

4.99

 

2

2.561

 

2.567

 

2.564

 

2.528

 

106.8

 

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, AD-464 does not possess an eye irritating potential.
Executive summary:

OECD 492 (2018) - This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.

Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer.

Tissues of the human cornea model EpiOcular™were treated with the test item, the positive and the negative control for 30 minutes each in duplicate.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 46.58%, thus the validity of the test system is ensured.

Relevant irritating effects were not observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 104.35 % compared with the value of the negative control (threshold for irritancy: ≤ 60%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess an eye irritating potential.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June 2018 - 29 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted to GLP and standard giudeline with all criteria met
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017
Deviations:
yes
Remarks:
Deviations did not affect the outcome of the assay.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: undiluted
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: no
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Freshly isolated bovine cornea obtained from AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals: Not reported/relevant - 3 Cornae per group
- Characteristics of donor animals (e.g. age, sex, weight): At least 9-month-old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were removed after slaughter, the isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: Same day
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) added to Styrofoam box during transportation.
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 (undiluted)
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
N/A
Duration of post- treatment incubation (in vitro):
210 Minutes (total) (opacity determination = 120 mins; permeability determination = 90 mins)
Number of animals or in vitro replicates:
3
Details on study design:
APPLICATION DOSE AND EXPOSURE TIME

0.75 mL applied to each cornea and incubated at 32 ± 1 °C in the water-bath for 10 minutes (± 30 seconds)

TREATMENT METHOD

Closed chamber

POST-INCUBATION PERIOD

Following rinsing, the corneas were incubated at 32 ± 1 °C (vertically) for 120 minutes followed by a second opacity reading (t130). The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea. In the second step of the assay, permeability of the corneae was determined. The permeability measurement was measured by optical density. The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red .

- POST-EXPOSURE INCUBATION:
Following rinsing, the corneas were incubated (vertically) for 120 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometer (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify) N/A

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As described in OECD 437.
Irritation parameter:
in vitro irritation score
Value:
8.56
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.
The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/UN GHS (Cat 1)). The mean IVIS of 110.71 was slightly above the 95% control limit of the Historical Control Data Base (64.24-110.03). This shows the sensitivity of the test and has no impact on the outcome if the study.

Table 1. Results after 10 Minutes Treatment Time

Test Group

Opacity value = Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

 

IVIS

Mean IVIS

Proposed

in vitro

Irritancy Score

Replicates

Mean

 

Replicates

Mean

 

 

Negative Control

 

0

0.33

0.060

0.065

0.90

1.31

No Category

1

0.065

1.98

0

0.071

1.07

Positive Control

83.67*

1.405*

 

104.74

110.71

Category 1

92.67*

1.432*

 

114.14

91.67*

1.439*

 

113.25

AD-464

7.67*

0.059*

 

8.55

8.56

No prediction can be made

8.67*

0.081*

 

9.88

6.67

0.039*

 

7.25

Interpretation of results:
study cannot be used for classification
Conclusions:
It was concluded that under the condition of this study, the test item, produced an IVIS score of 8.56 and therefore, no prediction for the damage hazard of the test item to the eye can be made in accordance with UN GHS/EU CLP Regulation (EC) 1272/2008.
Executive summary:

OECD 437 (2018) -The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using test item in accordance with OECD Guideline 437 "Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage" (2017).

Undiluted test item of 0.75 mL volume was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared using a similar concentration and volume of the test item . After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another app. 2 hours at 32 ± 1 °C in incubation medium, and opacity was measured for a second time (t130).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.31).

The mean IVIS of positive control was  110.71, slightly above the 95% control limit of the Historical Control Data Base (64.24-110.03). This shows the sensitivity of the test and has no impact on the outcome if the study.

The calculated mean IVIS was 8.56 (threshold for serious eye damage: IVIS > 55).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion/irritation

OECD 439 (2018): After treatment with the test item the mean relative viability value decreased to 97.78% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Eye corrosion/irritation

OECD 437 (2018): The calculated mean IVIS was 8.56 (threshold for serious eye damage: IVIS > 55).

OECD 492 (2018): Relevant irritating effects were not observed following 30 minutes incubation with the test item.

The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 104.35 % compared with the value of the negative control (threshold for irritancy: ≤ 60%).

Justification for classification or non-classification

During the OECD 439 study mean relative viability value decreased to 97.78 %, compared to negative controls. Therefore, the test item is not considered to possess an irritant potential. In accordance with GHS and Regulation (EC) No 1272/2008 (CLP) the study is not considering to be an irratant or corrosive to the skin.

During the OECD 437 study, the mean IVIS value was 8.56. Under the condition of the study and according to according to UN GHS/EU Regulation (EC) No. 1272/2008 regulation no prediction for the damage hazard of the test item to the eye can be made. Therefore a OECD 492 study was conducted.

During the OECD 492 study, the mean relative absorption value of the tissues corresponding to the cornea viability decreased to 104.35 % compared with the value of the negative control (threshold for irritancy: ≤ 60%).

The studies indicate that the substance does not meet the criteria for eye irritation or damage classification in accordance with GHS and Regulation (EC) No 1272/2008 (CLP).