Reaction mass of 5,7-dimethoxy-3-(4-(sec-C10-C13-alkyl)-benzoyl)-2H-chromen-2-one

Endpoint summary

Description of key information

Studies conducted to recognised testing guidelines with GLP certification

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Feb 2019 to 08 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Light yellow solid
Purity/Composition: UVCB (100%)
Storage Conditions: At room temperature protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: 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:

Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item:
Esacure 3644 was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, at least 25 mg of the test item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

Test for Reduction of MTT by the Test Item:
Esacure 3644 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 mg of the test item or 50 µL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.

Test System Set Up:
Tissues:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM.


Figure 1: A Diagram of the Application (attached below)

DMEM (Dulbecco’s Modified Eagle’s Medium):
Supplemented DMEM, serum-free supplied by MatTek Corporation.

MTT medium:
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions:
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 62 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.2 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Item Preparation:
No correction was made for the purity/composition of the test item. The solid test item was applied directly on top of the skin tissue. Esacure 3644 was spread to match the size of the tissue.

Application/Treatment of the Test Item:
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue (see figure 1). The plates were incubated for approximately 1 hour at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before Esacure 3644 was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to Esacure 3644 and two for a 1-hour exposure. The skin was moistened with 25 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 27.6 to 32.1 mg of the solid test item was added into the 6-well plates on top of the skin tissues.

For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data.

After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

Cell Viability Measurement:
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The solid test item was applied undiluted (27.6 to 32.1 mg) directly on top of the tissue.

Duration of treatment / exposure:

3 minutes for two tissues
1 hour for two other tissues

Duration of post-treatment incubation (if applicable):

The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2.

Number of replicates:

2 replicates for each exposure time

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean tissue viability, percentage of control

Run / experiment:

3-minute application

Value:

87

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

29

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean tissue viability, percentage of control

Run / experiment:

1-hour application

Value:

77

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

13

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Esacure 3644 was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with Esacure 3644 and controls are presented in Appendix 1, Table 1. The individual OD570 measurements are presented in Appendix 2.

Table 2 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with Esacure 3644 compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Esacure 3644 compared to the negative control tissues was 87% and 77% respectively. Because the mean relative tissue viability for Esacure 3644 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment Esacure 3644 is considered to be not corrosive.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range (See Appendix 3). The mean relative tissue viability following the 1-hour exposure to the positive control was 13%.

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤14% for the negative control and test item (Appendix 1, Table 3). For the positive control, the Coefficient of Variation between tissue replicates at the 3-minute exposure was 36%. Since both individual tissues were clearly positive it was concluded that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Esacure 3644 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate Esacure 3644 for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of Esacure 3644 was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch M7-238-1807001 of Esacure 3644 was a light yellow solid. Skin tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of Esacure 3644 was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 13% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤14% for the negative control and test item. For the positive control, the Coefficient of Variation between tissue replicates at the 3-minute exposure was 36%. Since both individual tissues were clearly positive it was concluded that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Esacure 3644 compared to the negative control tissues was 87% and 77%, respectively. Because the mean relative tissue viability for Esacure 3644 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Esacure 3644 is considered to be not corrosive.

In conclusion, Esacure 3644 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2019 - 18 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

EC Guideline No. 440/2008. Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Esacure 3644
Physical Description: Light yellow solid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from a single donor

Details on animal used as source of test system:

Adult human donor

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin
- Tissue batch number(s): 19-EKIN-011
- Date of initiation of testing: 13 March 2019

TEST SYSTEM SET UP
On the day of receipt, the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 23 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT MEDIUM
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 71 - 95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

TEST ITEM PREPARATION
No correction was made for the purity/composition of the test item. The solid test item was applied directly on top of the skin tissue. Esacure 3644 was spread to match the size of the tissue.

APPLICATION/TREATMENT OF THE TEST ITEM
The test was performed on a total of 3 tissues per test item together with negative and positive controls. The skin was moistened with 5 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (13.35 to 21.88 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 72 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

13.35 to 21.88 mg

Duration of treatment / exposure:

exposure period of 15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

The tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

3 tissues per test item together with negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

104

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:

Esacure 3644 was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20186172).
Because no color changes were observed it was concluded that Esacure 3644 did not interact with the MTT endpoint.
Since the mean relative tissue viability for Esacure 3644 was above 50% after 15 ± 0.5 minutes treatment Esacure 3644 is considered to be non-irritant.

[Appendicies 1 -3 containing all results data are attached to the background material section]

Esacure 3644 was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20186172).   Because no color changes were observed it was concluded that Esacure 3644 did not interact with the MTT endpoint.  

The mean absorption at 570 nm measured after treatment with Esacure 3644 and controls are presented in Appendix 1, Table 1.

The individual OD570 measurements are presented in Appendix 2.

Table 2 shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with Esacure 3644 compared to the negative control tissues.  Skin irritation is expressed as the remaining cell viability after exposure to the test item.  The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Esacure 3644 compared to the negative control tissues was 104%.  Since the mean relative tissue viability for Esacure 3644 was above 50% Esacure 3644 is considered to be non-irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 6.1%.  The absolute mean OD₅₇₀ of the negative control tissues was within the laboratory historical control data range (See Appendix 3).  The standard deviation value of the percentage viability of three tissues treated identically was < 6%, indicating that the test system functioned

properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Esacure 3644 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate Esacure 3644 for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)).  The possible skin irritation potential of Esacure 3644 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch M7-238-1807001 of Esacure 3644 was a light yellow solid.  Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of Esacure 3644 was applied directly on top of the skin tissue for 15 ± 0.5 minutes.  After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed.  Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item.  The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Esacure 3644 compared to the negative control tissues was 104%.  Since the mean relative tissue viability for Esacure 3644 was above 50% after 15 ± 0.5 minutes treatment Esacure 3644 is considered to be non-irritant.

The positive control had a mean cell viability of 6.1% after 15 ± 0.5 minutes exposure.  The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.  The standard deviation value of the percentage viability of three tissues treated identically was < 6%, indicating that the test system functioned properly.

In conclusion, Esacure 3644 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Feb 2019 - 26 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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:

adopted October 09, 2017

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

dentification: Esacure 3644
Physical Description: Light yellow solid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Bovine eyes were used as soon as possible after slaughter.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Characteristics of donor animals: Young cattle obtained from the slaughterhouse

PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The
anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

315.0 to 354.3 mg, applied directly on the corneas in such a way that the cornea was completely covered

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C

Duration of post- treatment incubation (in vitro):

After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure Uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item
on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

-0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

-0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: Permeability

Run / experiment:

Mean

Value:

0.034

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:

ACCEPTANCE OF RESULTS:
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 (20% (w/v) Imidazole) was 139 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Esacure 3644 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.1 after 240 minutes of treatment.

Summary

Treatment	Mean Opacity	Mean Permeability	Mean in vitro irritation score (1,2)
Negative control	0.8	0.014	1
Positive control	117	1.465	139
Esacure 3644	-0.6	0.034	-0.1

(1) Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.

(2) In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).7

Individual results are detailed in the full study report

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since Esacure 3644 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Esacure 3644 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas.  The eye damage of Esacure 3644 was tested through topical application for approximately 240 minutes.  

The study procedures described in this report were based on the most recent OECD guideline.

Batch M7-238-1807001 of Esacure 3644 was a light yellow solid.  Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.

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 (20% (w/v) Imidazole) was 139 and within two standard deviations of the current historical positive control mean.  It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

Esacure 3644 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.1 after 4 hours of treatment.  

In conclusion, since Esacure 3644 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Substance does not fulfil the CLP criteria for classification against this endpoint.