2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

Endpoint summary

Description of key information

Skin Irritation / Corrosion

Skin corrosion in vitro. Key CRL 2019

OECD 431

The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Skin irritation in vitro. Key CRL 2020

OECD 439

The test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Skin irritation / corrosion in vivo. Key CRL 2020

OECD 404

Based on these results the test item does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Eye Irritation

Eye irritation in vitro. Key CRL 2019

OECD 437

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

Eye irritation in vivo. Key CRL 2020

OECD 405

Based on these results, the test item does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments). 

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:

14 November 2019 - 28 January 2020

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:

adopted 18 June 2019

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:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

Test system:

human skin model

Remarks:

EpiDerm Skin Model (EPI-200, Lot no.: 32126 Kit A and kit B)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: All cells used to produce EpiDerm are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Model used: EpiDerm Skin Model (EPI-200)
Tissue batch number: Lot no.: 32126 Kit A and kit B
Production date: November 2019
Date of initiation of testing: 25 November 2019
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.
DMEM (Dulbecco’s Modified Eagle’s Medium) - Supplemented DMEM, serum-free 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 63 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°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.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

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
The test item 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, 50 µL 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
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µL 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 ITEM PREPARATION/APPLICATION
No correction was made for the purity/composition of the test item.
The liquid test item was applied undiluted (50 µL) directly on top of the tissue.

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 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item 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 the test item and two for a 1-hour exposure.

Fifty µL of the undiluted 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 (see deviation) 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.


ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:

a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8).
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

No correction was made for the purity/composition of the test item.
The liquid test item was applied undiluted (50 µL) directly on top of the tissue.

Duration of treatment / exposure:

The test consists of topical application of the test item on the skin tissue for 3-minute and 1-hour.

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application viability (percentage of control)

Value:

101

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application viablilty (percentage of control)

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item 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 the test item and controls are presented below (and in appendix 1, Table 1 in attached background material):

Mean Absorption in the in vitro Skin Corrosion Test with the test item

	3 minute application					1 hour application
	A (OD570)	B (OD570)	Mean(OD570)	SD		A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	1.643	1.773	1.708	±	0.092	1.643	1.773	1.708	±	0.092
Test item	1.786	1.674	1.73	±	0.079	1.786	1.674	1.73	±	0.079
Positive control	0.288	0.368	0.328	±	0.056	0.288	0.368	0.328	±	0.056

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.

The individual OD570 measurements are presented in Appendix 2 (in attached backrgound material).  

The below table (appendix 1, table 2 in attached background material) shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test item 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 the test item compared to the negative control tissues was 101% and 98% respectively.  Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

Mean Tissue Viability in the in vitro Skin Corrosion Test with C991

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	101	98
Positive control	19	7.3

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 (Appendix 3 in attached background material). The mean relative tissue viability following the 1-hour exposure to the positive control was 7.3%.  

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 8%, indicating that the test system functioned properly (below table, appendix 1, table 3 in attached background material).

Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	7.3	5.6
Test item	6.3	4.1
Positive control	22	8

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Interpretation of results:

GHS criteria not met

Conclusions:

2 -ethylhexyl 2 -{[1,1'-biphenyl]-4 -carbonyl}benzoate 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 the test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item 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.

The test item was a colourless to pale yellow liquid. The test item was applied undiluted (50 µL) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 7.3% 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 < 8%, indicating 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 the test item compared to the negative control tissues was 101% and 98%, respectively. 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 1-hour treatment the test item is considered to be not corrosive.

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

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2020 - 24 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines

Version / remarks:

2000 & including the most recent revisions

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name: 2-Ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate
CAS number: 75005-95-7
EC Number: 278-051-5
Molecular formula: C28H30O3
Molecular weight: 414.22
pH (1% in water, indicative range): 6.36 – 6.58 (determined by Charles River Den Bosch)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST SYSTEM
Species: Rabbit
Strain: New Zealand White
Condition: SPF-Quality
Source: Charles River France, L’Arbresle, France
Number of Animals: 3 Males.
Age at the Initiation of Dosing: Young adult animals (approximately 11-22 weeks old) were selected.
Weight at the Initiation of Dosing: 2383 to 3788 g.

Animal Identification
At study assignment, each animal was identified using an ear mark with indelible ink.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study. Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

HUSBANDRY
Housing
On arrival and following assignment to the study, animals were housed individually in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. The room in which the animals were kept was documented in the study records. Each cage was clearly labeled.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 18-19°C with an actual daily mean relative humidity of 54 to 55%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Granovit AG, Kaiseraugst, Swizerland) was provided once daily throughout the study. In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was available during the study period. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test
Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the
Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm) and wooden sticks (Swedish aspen
wood, Bioservices, Uden, The Netherlands) except when interrupted by study procedures/activities.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

Each animal was treated by dermal application of 0.5 mL of the test item. The test item was applied to the skin of one flank, using a metalline patch of 2x3 cm

Duration of treatment / exposure:

Four hours

Observation period:

The skin reactions were assessed at approximately 1, 24, 48 and 72 hours and 7 days after the removal of the dressings and test item.

Number of animals:

3 Males

Details on study design:

In-life Procedures, Observations, and Measurements

Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Toxicity
Observations for toxicity were performed once daily throughout the study.

Body Weights
Animals were weighed individually on Day 1 (pre-dose) and on the day of the final observation.

Irritation
The skin reactions were assessed at approximately 1, 24, 48 and 72 hours and 7 days after the removal of the dressings and test item. The irritation scores and a description of all other
(local) effects were recorded. Adjacent areas of the untreated skin of each animal served as controls.



The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

Erythema and eschar formation:
No erythema ............................................................................................................................................................0
Very slight erythema (barely perceptible) ...............................................................................................................1
Well-defined erythema ............................................................................................................................................ 2
Moderate to severe erythema ................................................................................................................................. 3
Severe erythema (beef redness) * ..........................................................................................................................4
*. Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema (= 4) is given.

Oedema formation:
No oedema .............................................................................................................................................................0
Very slight oedema (barely perceptible) ................................................................................................................1
Slight oedema (edges of area well-defined by definite raising) ............................................................................2
Moderate oedema (raised approximately 1 millimeter) ........................................................................................... 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) ............................. 4

Histopathology
No histopathology was performed since sufficient information was obtained within this study.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of weak irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of weak irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of weak irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Irritation
Four hours exposure to 0.5 mL of C991 resulted in very slight erythema in the treated skin areas of the three rabbits. The skin irritation resolved within 7 days after exposure in all animals. Table 1 shows individual results below.

Other effects:

Coloration / Remnants
Remnants of the test item were present on the skin of one animal on Day 1 and two animals on Days 1 and 2.

Toxicity / Mortality
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Table 1 - Individual Skin Irritation Scores

Animal	480 (1)			486			487
Time after exposure	Erythema (0-4)	Oedema (0-4)	Comments	Erythema (0-4)	Oedema (0-4)	Comments	Erythema (0-4)	Oedema (0-4)	Comments
1 hour	1	0	a	1	0	a	1	0	a
24 hours	1	0	b	1	0	b	1	0	-
48 hours	1	0	-	1	0	-	1	0	-
72 hours	1	0	-	1	0	-	1	0	-
7 days	0	0	-	0	0	-	0	0	-

1 Sentinel.

Comments:

a. Sticky remnants of the test item present.

b. Greasy remnants of the test item present.

- No comments

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results the test item does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this primary skin irritation study was to assess the possible irritation or corrosion potential of a single dose of the test item when administered to the intact skin of rabbits.

The study was carried out in compliance with the guidelines described in:

• OECD No. 404 (2015) "Acute Dermal Irritation / Corrosion".

• EC No 440/2008, part B: "Acute Toxicity: Dermal Irritation/Corrosion".

• EPA, OPPTS 870.2500 (1998), "Acute Dermal Irritation".

• JMAFF Guidelines (2000), including the most recent revisions.

Three rabbits were exposed to 0.5 mL of the test item by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours and 7 days after exposure. Exposure to the test item resulted in very slight erythema in the treated skin areas of all rabbits, which had resolved within 7 days. Remnants of the test item were present on the skin of one animal on Day 1 and two animals on Days 1 and 2.

Based on these results the test item does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 December 2019 - 27 February 2020

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:

adopted 18 June 2019

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.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test". Official Journal of the European Union No. L142

Version / remarks:

Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Deviations:

no

Principles of method if other than guideline:

Deviation:
Cell Viability Measurement
The study plan states that extraction with isopropanol should be for 70 +/- 3 hours. However, the extraction was performed for 66 hours, which is not according to the study plan.

Evaluation: According to Episkin validation model, formazan extraction should be performed for 18 to 72 hours. In addition, all criteria are met. Therefore, the assay was still valid and the 66 hours extraction did not influence the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Test item handling: No specific handling conditions required
CAS number: 75005-95-7
EC Number: 278-051-5
Substance Name: 2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

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 Small ModelTM (EPISKIN-SMTM, 0.38 cm2,
Tissue batch number: Batch no.: 20 EKIN 002
Production date: January 2020
Date of initiation of testing: 07 January 2020
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 23 hours at 37°C (Figure 1). Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.


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 65 - 95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.9 - 37.4°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.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).

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. The test item 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. 20221673). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

TEST ITEM PREPARATION AND CONDITIONS
No correction was made for the purity/composition of the test item.
The liquid test item was applied undiluted (25 µL) directly on top of the tissue.

The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five µL of the undiluted test item 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-labeled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 66 hours (see Study plan deviation). The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit ≤1.5) and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Twenty five µL of the undiluted test item 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.

No correction was made for the purity/composition of the test item.
The liquid test item was applied undiluted (25 µL) directly on top of the tissue.

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value

Value:

97

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:

The test item 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. 20221673). Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with the test item and controls are presented in the table below (and attached in Appendix 1, Table 1).

Table 1 - Mean Absorption in the In Vitro Skin Irritation Test with C991

	A	B	C	Mean		SD
	(OD570)	(OD570)	(OD570)	(OD570)
Negative control	1.1087	1.0596	1.121	1.0964	±	0.0325
Test item	1.0573	0.9742	0.8727	0.9681	±	0.0924
Positive control	0.0526	0.1149	0.0902	0.0859	±	0.0313

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.046). Isopropanol was used to measure the background absorption.

The individual OD570 measurements are presented in Appendix 2 (in attached background material).

Table 2 below (and in attached background material, Appendix 1) shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item 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 the test item compared to the negative control tissues was 97%.  Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

Table 2 - Mean Tissue Viability in the In Vitro Skin Irritation Test with C991

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	3
Test item	97	9
Positive control	7.8	3

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.8%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Appendix 3 in attached background material). The standard deviation value of the percentage viability of three tissues treated identically was ≤ 9%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the test tiem is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the test item 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 the test item 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.

The test item was a colourless to pale yellow liquid with a purity of 96.27%. The test item was applied undiluted (25 µL), 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 the test item compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 7.8% 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 ≤ 9%, indicating that the test system functioned properly.

In conclusion, the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 2019 - 13 January 2020

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 (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
CAS number: 75005-95-7

Species:

cattle

Details on test animals or tissues and environmental conditions:

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.

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.

Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. 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) Foetal 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.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Cornea Selection and Opacity Reading: After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item (excessive amount) was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

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

Duration of post- treatment incubation (in vitro):

Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.

Number of animals or in vitro replicates:

3

Details on study design:

TEST ITEM PREPARATION
No correction was made for the purity/composition of the test item.
The test item was tested neat.


TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item (excessive amount) was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a
horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.
After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.


OPACITY MEASUREMENT
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:

Opacity = (( I0 / I ) - 0.9894) / 0.0251.

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was
calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.


APPLICATION OF SODIUM FLUORESCEIN
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 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. 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.


PERMEABILITY DETERMINATIONS
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

cornea opacity score

Remarks:

Mean Opacity

Run / experiment:

Test item

Value:

1.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Mean Permeability

Run / experiment:

Test item

Value:

0.025

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

Mean In vitro Irritation Score

Run / experiment:

Test item

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Remarks:

Mean Opacity

Run / experiment:

Negative control

Value:

2.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Mean Permeability

Run / experiment:

Negative control

Value:

0.003

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

Mean In vitro Irritation Score

Run / experiment:

Negative control

Value:

2.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Remarks:

Mean Opacity

Run / experiment:

Positive control (Ethanol)

Value:

20

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Mean Permeability

Run / experiment:

Positive control (Ethanol)

Value:

1.888

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

Mean In vitro Irritation Score

Run / experiment:

Positive control (Ethanol)

Value:

48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Results calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

The test item was tested neat.  

The above in-vitro data summarizes the opacity, permeability and in vitro irritancy scores of the test item and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2 - 5 in the attached appendix.  

The individual in vitro irritancy scores for the negative controls ranged from 2.4 to 2.9. The corneas treated with the negative control item were clear after the 10 minutes of treatment.

The individual positive control in vitro irritancy scores ranged from 40 to 54 (Appendix, Table 5).  The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from -0.8 to 4.0 and permeability values ranging from 0.009 to 0.057. The corneas were translucent/clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.  Hence, the in vitro irritancy scores ranged from 0.0 to 4.1 after 10 minutes of treatment with the test item.

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 48 and within two standard deviations of the current historical positive control mean (Appendix, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.7 after 10 minutes of treatment. Although one of the test item treated corneas resulted in an IVIS score of 4.1, the IVIS score was only slightly above the cut-off value of 3 and was close to the mean IVIS score of 1.7.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since the test item 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 the test item 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 the test item was tested through topical application for 10 minutes.

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

The test item was a colourless to pale yellow liquid with a purity of 96.27%. The test item was applied as it is (excessive amount) directly 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 (Ethanol) was 48 and was 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.

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.7 after 10 minutes of treatment.

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

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Mar 2020 - 06 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines

Version / remarks:

2000, including the most recent revisions

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name: 2-Ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate
CAS number: 75005-95-7
EC Number: 278-051-5
Molecular formula: C28H30O3
Molecular weight: 414.22

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species: Rabbit
Strain: New Zealand White
Condition: SPF-Quality
Source: Charles River France, L’Arbresle, France
Number of Animals: 3 Males.
Age at the Initiation of Dosing: Young adult animals (approximately 12-14 weeks old) were selected.
Weight at the Initiation of Dosing: 2376 to 3077 g.

Justification for Test System and Number of Animals
The New Zealand White rabbit was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals were based on the test guidelines.
The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified using an ear mark with indelible ink.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
The disposition of all animals was documented in the study records.

Husbandry
Housing
On arrival and following assignment to the study, animals were housed individually in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. The room in which the animals were kept was documented in the study records. Each cage was clearly labeled.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19°C with an actual daily mean relative humidity of 51 to 54%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Granovit AG, Kaiseraugst, Swizerland) was provided once daily throughout the study. In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was available during the study period.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.


Study Initiation Date: 25 Mar 2020
Initiation of Dosing: 30 Mar 2020
Completion of In-life: 10 Apr 2020
Experimental Start Date: 30 Mar 2020
Experimental Completion Date: 10 Apr 2020

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

0.1 mL of the test item

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 males

Details on study design:

Experimental Design
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 1 week later, after considering the degree of eye irritation observed in the first animal.

Pre-emptive Pain Management
One hour prior to instillation of the test item, buprenorphine (Buprenodale®) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test item, two drops of the topical anaesthetic 0.5% proparacaine hydrochloric ophthalmic solution (Tetracaine eye drops®) were applied to both eyes.

Administration of Test item
Each animal was treated by instillation of 0.1 mL of the test item, in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test item. The other eye remained untreated and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage.
In order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection.

Justification of Route and Dose Level
The ocular route was selected because the test item may accidentally come into contact with the eyes during manufacture, handling and/or use. The dose level was based on the test guidelines.

In-life Procedures, Observations, and Measurements
Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Toxicity
Observations for toxicity were performed once daily throughout the study.

Body Weights
Animals were weighed individually on Day 1 (pre-dose) and on the day of the final observation.

Irritation (see table below)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

Irritation
Instillation of 0.1 mL of the test item into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness and discharge. The irritation had completely resolved within 48 hours in all animals.
No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test item instillation revealed no corneal epithelial damage.

Coloration / Remnants
No staining of (peri) ocular tissues by the test item was observed and no test item remnants were seen.

Toxicity / Mortality
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results, the test item does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this acute eye irritation study was to assess the possible irritation or corrosion potential when a single dose of the test item was placed in the conjunctival sac of the rabbit eye.

The study was carried out in compliance with the guidelines described in:

- OECD No.405 (2017) "Acute Eye Irritation / Corrosion".

- EC No 440/2008, part B: "Acute Toxicity: Eye Irritation/Corrosion".

- EPA, OPPTS 870.2400 (1998), "Acute Eye Irritation".

- JMAFF Guidelines (2000), including the most recent revisions.

Single samples of 0.1 mL of the test item were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours after instillation.

Instillation of the test item resulted in irritation of the conjunctivae, which consisted of redness and discharge. The irritation had completely resolved within 48 hours in all animals.

Based on these results, the test item does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Studies conducted to recognised testing guidelines with GLP certification.

Justification for classification or non-classification

Skin Irritation / Corrosion

Based on these results, the test item does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Eye Irritation

Based on these results, the test item does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).