[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin irritation - In conclusion, due to the properties of the test material, Red HF2 was deemed not fit for this in vitro skin irritation test.

 

Eye irritation - Since 3 out of 6 corneas were spread over 2 categories (IVIS <3 and > 3 ≤ 55), the test is inconclusive.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August - 29 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study conducted to current guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details in the study report.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Human

Source strain:

other: Human

Details on animal used as source of test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 22 EKIN 034.
This model is a three-dimensional human epidermis model, which consists of adult humanderived
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.

Source
EPISKIN, Lyon, France.

Justification for test system used:

Rationale
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:

Test system
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 22 EKIN 034.
This model is a three-dimensional human epidermis model, which consists of adult humanderived
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.


Source
EPISKIN, Lyon, France.
Experimental Design
Test for the Interference of the Test Material with the MTT Endpoint
A test material 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 material is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Material
The test material was checked for possible color interference before the study was started.
Some non-colored test materials may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, approximately 50 mg of the test material or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, approximately 50 mg of the test material or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking.
At the end of the exposure time, the mixtures were centrifuged for 30 seconds at 16000 g of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
If after subtraction of the negative control, the OD for the test material solution is >0.08, the test material is considered as possibly interacting with the MTT measurement.

Test for Reduction of MTT by the Test Material
The test material was checked for possible direct MTT reduction before the study was started.
To assess the ability of the test material to reduce MTT, approximately 50 mg of the test material was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 μL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test material interacts with MTT.

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 EPISKIN, Lyon, France.
Killed tissues (EPISKIN-SMTM, 0.38 cm2, Lot no.: 22 EKIN 013, 22 EKIN 030.
Living epidermis was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored in a freezer set to maintain -15°C.
Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 mL maintenance medium. Further use of killed tissues was similar to living tissues.
MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 1 mg/mL in PBS) diluted (3x) in Assay medium (final concentration 0.3 mg/mL).
Environmental conditions
All incubations, with the exception of the test material 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 44 - 97%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 - 37.0°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 Material Preparation
The solid test material liquified by heating until 65.0 °C in order to homogenize and weigh the test material. Afterwards, the test material remained in a very viscous paste-like state. The test material could therefore not be crushed and ground using pestle and mortar and was treated like a paste. Prior to treatment the test material was heated again to 60.0 °C and placed on a nylon mesh. The test material was then applied directly on top of the skin tissue.

Application/Treatment of the Test Material
The test was performed on a total of 3 tissues per test material 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 material to the tissue and the solid test material was added in excess amount into 12-well plates on top of the skin tissues, using a nylon mesh. The test material was difficult to apply since it remained stuck to the mesh. 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. Since the test material reacted with the MTT medium, in addition three killed tissues treated with test material and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. Since the test material showed color interference in aqueous conditions, in addition to the normal procedure, three tissues were treated with test material. Instead of MTT solution these tissues were incubated with assay medium. Furthermore, since the test material was identified as MTT reducer and caused color interference, the test material was applied to three killed tissue replicates which underwent the entire testing procedure but were incubated
with assay medium instead of MTT solution during the MTT assay. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test material. The test material was difficult to remove, as it remained stuck to the skin and the insert. 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 ± 1 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 hours ± 5 minutes 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 approximately 69 hours. Extractants were moved to a 96-wells plate. In several wells the isopropanol was colored red. 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:

To ensure close contact of the test material to the tissue and the solid test material was added in excess amount into 12-well plates on top of the skin tissues, using a nylon mesh. The test material was difficult to apply since it remained stuck to the mesh. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 ± 1 hours at 37°C.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

due to the properties of the test material, Red HF2 was deemed not fit for this in vitro skin irritation test.

Other effects / acceptance of results:

The test material was checked for color interference in aqueous conditions. Addition of the test material to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0014 and ≥4.0 (overflow), respectively. Therefore, it was concluded that the test material induced color interference.
Because a color change was observed in the presence of MTT it was concluded that the test
material interacted with the MTT endpoint.
The individual OD570 measurements are presented in any other information on results.
The mean absorption at 570 nm measured after treatment with the controls are presented in
any other information on results.
Table 2 (see any other information on results) shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with the positive control compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test material, The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.9%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Table 4 in any other information on results) and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit ≤1.5). The standard deviation value of the percentage viability of three tissues treated identically with positive and negative controls was ≤ 2.3%, indicating that the test system functioned properly.
During treatment with test material, it was noted that the test material was very viscous and was difficult to apply to the tissues, since it remained stuck to the nylon mesh. As a result, the tissues might not have been completely covered. In addition, the test material was difficult to remove from the tissues and inserts. As a result, any remaining test material colored the isopropanol red, which might have interfered with the measurements. These properties have interfered with the experiment, giving unreliable results. In addition to the normal procedure, three tissues were treated with test material. Instead of MTT solution these tissues were incubated with assay medium. For one of the three tissues an ‘overflow’ was measured, which equals to an OD ≥ 4.0. As a result, the non-specific color by the test material would be ≥121% of the negative control tissues, which does exceed the acceptability criteria and the results. This high measurement, similar to in the test for color interference, is caused by residual test material, which also colored the isopropanol red. In addition to the normal procedure, three killed tissues treated with test material and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The nonspecific reduction of MTT by the test material was -2.8% of the negative control tissues.
Since the %NSMTT was below 0.0, there was no need to correct for MTT interference. Furthermore, since the test material was identified as MTT reducer and caused color interference, the test material was applied to three killed tissue replicates which underwent the entire testing procedure but were incubated with assay medium instead of MTT solution during the MTT assay. Since the %NSMTT was below 0.0, there was no need to correct for the non-specific color interference on killed tissues (NSCkilled).
Due to the properties of the test material, the high color interference, and the red-colored isopropanol, the relative mean tissue viability after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was not reliable and, therefore, not reported.

Table 1

Mean Absorption in the In Vitro Skin Irritation Test with Red HF2

 

	A	B	C	Mean		SD
	(OD570)	(OD570)	(OD570)	(OD570)
Negative control	1.139	1.088	1.116	1.114	±	0.026
Test item	(1)	(1)	(1)	(1)	±	(1)
Positive control	0.104	0.062	0.098	0.088	±	0.023

OD = optical density SD = Standard deviation

(1)  Not reported since the results are unreliable, due to the properties of the test material, high

%NSCliving (color interference), and red isopropanol after formazan extraction. Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.044). Isopropanol was used to measure the background absorption.

 

Table 2

Mean Tissue Viability in the In Vitro Skin Irritation Test with Red HF2

 

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	2.3
Test item	(1)	(1)
Positive control	7.9	2.1

(1)  Not reported since the results are unreliable, due to the properties of the test material, high

%NSCliving (color interference), and red isopropanol after formazan extraction.

Table 3

Individual OD Measurements at 570 nm

 

	A (OD570)	B (OD570)	C (OD570)
Negative control
OD570 measurement 1	1.1865	1.1390	1.1645
OD570 measurement 2	1.1792	1.1247	1.1547
Test material on viable tissue
OD570 measurement 1	0.2884 ^(1)	0.2511 ^(1)	1.0685
OD570 measurement 2	0.2837 ^(1)	0.2699 ^(1)	1.0566
Test material on viable tissue
(no MTT)
OD570 measurement 1	0.1255	0.0557	Overflow ^(1)
OD570 measurement 2	0.1257	0.0573	Overflow ^(1)
Non treated killed tissue
OD570 measurement 1	0.1133	0.1108	0.1186
OD570 measurement 2	0.1121	0.1069	0.1214
Treated killed tissue
OD570 measurement 1	0.0985	0.0682	0.0742
OD570 measurement 2	0.1000	0.0785	0.0737
Treated killed tissue (no MTT)
OD570 measurement 1	0.0854 ^(1)	0.0496	0.0866 ^(1)
OD570 measurement 2	0.0826 ^(1)	0.0489	0.0873 ^(1)
Positive control
OD570 measurement 1	0.1479	0.1038	0.1431
OD570 measurement 2	0.1481	0.1073	0.1409

⁽¹⁾ isopropanol was colored red after formazan extraction. OD = Optical density

Overflow = OD ≥ 4.0

Triplicate exposures are indicated by A, B and C.

Table 4

Historical Control Data for In Vitro Skin Irritation Studies

 

 

	Negative control (absorption; OD570)	Positive control (absorption; OD570)
Min	0.507	0.021
Max	1.478	0.549
Mean	1.090	0.097
SD	0.164	0.077
n	177	177

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2019 to May 2022.

 

Interpretation of results:

other: Inconclusive

Conclusions:

In conclusion, due to the properties of the test material, Red HF2 was deemed not fit for this in vitro skin irritation test.

Executive summary:

The objective of this study was to evaluate Red HF2 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 material 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 164:1 of the test material was a dark red solid. The test material was applied undiluted The solid test material liquified by heating until 65.0 °C in order to homogenize and weigh the test material. Afterwards, the test material remained in a paste-like state. Prior to treatment the test material was heated again to 60.0 °C and an excess amount was placed on a nylon mesh. Skin tissue was moistened with 5 μL of Milli-Q water and the test material was then applied directly on top of the skin tissue for 15 ± 0.5 minutes.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.9%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and
upper acceptance limit 1.5). The standard deviation value of the percentage viability of three tissues treated identically with positive and negative controls was ≤ 2.3%, indicating that the test system functioned properly.
During treatment with test material, it was noted that the test material was very viscous and was difficult to apply to the tissues, since it remained stuck to the nylon mesh. As a result, the tissues might not have been completely covered. In addition, the test material was difficult to remove from the tissues and inserts. As a result, any remaining test material colored the isopropanol red, which might have interfered with the measurements. These properties have interfered with the experiment, giving unreliable results.
In addition to the normal procedure, three tissues were treated with test material. Instead of MTT solution these tissues were incubated with assay medium. For one of the three tissues an ‘overflow’ was measured, which equals to an OD ≥ 4.0. As a result, the non-specific color by
the test material would be ≥121% of the negative control tissues, which does exceed the acceptability criteria and the results. This high measurement, similar to in the test for color interference, is caused by residual test material, which also colored the isopropanol red.
In addition to the normal procedure, three killed tissues treated with test material and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The nonspecific reduction of MTT by the test material was -2.8% of the negative control tissues.
Since the %NSMTT was below 0.0, there was no need to correct for MTT interference.
Furthermore, since the test material was identified as MTT reducer and caused color interference, the test material was applied to three killed tissue replicates which underwent the entire testing procedure but were incubated with assay medium instead of MTT solution during the MTT assay. Since the %NSMTT was below 0.0, there was no need to correct for the non-specific color interference on killed tissues (NSCkilled).
Due to the properties of the test material, the high color interference, and the red-colored isopropanol, the relative mean tissue viability after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was not reliable and, therefore, not reported.
Results are retained within the raw data In conclusion, due to the properties of the test material, Red HF2 was deemed not fit for this in vitro skin irritation test.

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:

17 August - 20 September 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study conducted to current guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details in the study report

Species:

other: Bovine Eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used in the test as soon as possible after slaughter.
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.
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 and tested the day of arrival in the laboratory.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Since no workable suspension of the test material in physiological saline could be obtained,
the test material was used as delivered and added directly on top of the corneas.
750µL of the negative control or 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test material was heated (temperature range of 60-65°C) until melted to obtain a homogeneous test material, then the test material was weighed in a bottle and applied on a filter paper. The test material on the filter paper was allowed to cool down for approximately 30 minutes to room temperature. The room temperature is set to maintain 21°C. The filter paper with test material was applied on the cornea in such a way that the cornea was
completely covered with the test material. (maximum and minimum amounts per cornea ranging between 6058.7 and 2446.02 mg, respectively exp 1 and 2)

Duration of treatment / exposure:

240 ± 10 minutes at 32 ± 1°C

Duration of post- treatment incubation (in vitro):

Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32± 1°C.

Number of animals or in vitro replicates:

3 per test

Details on study design:

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 Duratec Analysentechnik GmbH (Hockenheim, 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.

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, Duratec GmbH). 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. Six corneas were selected at random for each treatment group.

Test Material Preparation
No correction was made for the purity/composition of the test material.
Since no workable suspension of the test material in physiological saline could be obtained, the test material was used as delivered and added directly on top of the corneas.

Treatment of Corneas and Opacity Measurements
The medium from the anterior compartment was removed and 750 µL of the negative control or 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test material was heated (temperature range of 60-65°C) until melted to obtain a homogeneous test material, then the test material was weighed in a bottle and applied on a filter paper. The test material on the filter paper was allowed to cool down for approximately 30 minutes to room temperature. The room temperature is set to maintain 21°C. The filter paper with test material was applied on the cornea in such a way that the cornea was completely covered with the test material. (maximum and minimum amounts per cornea ranging between 6058.7 and 2446.02 mg, respectively exp 1 and 2) The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240±10 minutes at 32 ± 1°C. After the incubation the solutions and the test material 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 material 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.

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 cornea treated with the test material 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 material 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 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.

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 material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
• The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
• The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
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.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

1.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

1.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

5.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2/1

Value:

6.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2/2

Value:

3.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2/3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: -0.2

Other effects / acceptance of results:

The test material was tested neat after test material was heated (temperature range of 60- 65°C) until melted to obtain a homogeneous test material, applied to filter paper and allowed to cool to room temperature .
Table 1 in any other information on results summarizes the mean opacity, permeability and in vitro irritancy scores (IVIS) of the test material and the controls. The opacity, permeability and IVIS of the individual corneas are shown in, Table 2 - 5 in any other information on results.
The individual IVIS for the negative controls ranged from 2.6 to 3.2. The corneas treated with the negative control material were clear after the 240 minutes of treatment. The individual positive control IVIS ranged from 122 to 153 (Table 5). The corneas treated with the positive control were turbid after the 240 minutes of treatment. No pH effects of the negative and positive control were observed on the rinsing medium.
The corneas treated with the test material showed opacity values of 1.9, 1.6 and 5.5 and permeability values ranging from -0.003 to 0.001. The corneas were clear after the 240 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the IVIS were 1.9,1.6 and 5.5 after 240 minutes of treatment with the test material.
Since the results included a discordant IVIS for one of the 3 corneas (IVIS of 1.6, 1.9 and 5.5, respectively), and the IVIS was >10 from 55 (as per the ‘AND’ criteria of OECD 437) the test was considered inconclusive, and a repeat experiment was performed. In the second experiment, the individual IVIS for the negative controls ranged from -0.7 to 2.8. The corneas treated with the negative control material were clear after the 240 minutes of treatment. The individual positive control IVIS ranged from 109 to 139 (Table 5). The corneas treated with the positive control were turbid after the 240 minutes of treatment. No pH effects of the negative and positive control were observed on the rinsing medium. The corneas treated with the test material showed opacity values of -0.4, 3.1 and 6.9 and permeability values of -0.008, 0.002 and 0.013. The corneas were clear after the 240 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the mean IVIS were -0.2, 3.1 and 6.8 after 240 minutes of treatment with the test material.
One of the individual IVIS results was discordant with the mean IVIS (IVIS of -0.2, 3.1 and 6.8, respectively) and the discordant value was >10 IVIS units from 55, the test was considered to be inconclusive. A third experiment was considered unnecessary.

Table 1

Summary of Opacity, Permeability and In Vitro Scores

 

First experiment

 

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score ^1, ^2
Negative control	2.5	0.021	2.8
Positive control	112	1.666	137
Test material	3.0	-0.002	3.0

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

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

Second experiment

 

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score ^1, ^2
Negative control	0.6	0.012	0.8
Positive control	90	2.530	128
Test material	3.2	0.002	3.2

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

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

Table 2 Opacity Score

 

First experiment

 

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected Final Opacity ^2	Mean Final Opacity
Negative control	0.9	3.0	2.1		2.5
	1.5	4.4	2.9
	0.9	3.5	2.5
Positive control	2.8	101	99	96	112
	2.8	137	134	131
	2.6	113	111	108
Test material	2.9	7.4	4.5	2.0	3.0
	3.6	7.7	4.1	1.6
	1.4	9.4	8.0	5.5

Calculations are made without rounding off.

¹ Final Opacity = Opacity after treatment – Opacity before treatment.

² Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

 

Second experiment

 

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected Final Opacity ^2	Mean Final Opacity
Negative control	1.1	3.8	2.6		0.6
	0.4	-0.6	-1.0
	3.7	4.0	0.3
Positive control	3.2	107	103	103	90
	3.3	105	101	101
	-1.8	66	68	67
Test material	2.6	10	7.5	6.9	3.2
	3.7	7.5	3.7	3.1
	3.3	3.6	0.3	-0.4

Calculations are made without rounding off.

¹ Final Opacity = Opacity after treatment – Opacity before treatment.

² Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

Table 3

Permeability Score Individual Values (Uncorrected)

 

First experiment

 

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	0.029	0.031	0.045	0.035	0.035	0.021
	1	0.015	0.010	0.021	0.015	0.015
	1	0.012	0.017	0.013	0.014	0.014
Positive control	6	0.457	0.455	0.462	0.458	2.748
	6	0.266	0.261	0.264	0.264	1.582
	1	0.951	0.950	0.941	0.947	0.947
Test material	1	0.019	0.018	0.021	0.019	0.019
	1	0.017	0.022	0.016	0.018	0.018
	1	0.022	0.023	0.021	0.022	0.022

Calculations are made without rounding off.

 

Second experiment

 

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	0.009	0.010	0.013	0.011	0.011	0.012
	1	0.016	0.019	0.017	0.017	0.017
	1	0.009	0.009	0.009	0.009	0.009
Positive control	6	0.388	0.385	0.385	0.386	2.316
	6	0.445	0.441	0.440	0.442	2.652
	6	0.479	0.485	0.458	0.474	2.844
Test material	1	0.004	0.004	0.004	0.004	0.004
	1	0.015	0.014	0.014	0.014	0.014
	1	0.025	0.026	0.025	0.025	0.025

Calculations are made without rounding off.

 

Table 4

Permeability Score Individual Values (Corrected)

 

First experiment

 

Treatment	Dilution factor	Negative control corrected OD490 1^1	Negative control corrected OD490 2^1	Negative control corrected OD490 3^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	6	0.436	0.434	0.441	0.437	2.619	1.666
	6	0.245	0.240	0.243	0.242	1.453
	1	0.930	0.929	0.920	0.926	0.926
Test material	1	-0.002	-0.003	0.000	-0.002	-0.002	-0.002
	1	-0.004	0.001	-0.005	-0.003	-0.003
	1	0.001	0.002	0.000	0.001	0.001

Calculations are made without rounding off.

¹ OD490 values corrected for the mean final negative control permeability

 

Second experiment

 

Treatment	Dilution factor	Negative control corrected OD490 1^1	Negative control corrected OD490 2^1	Negative control corrected OD490 3^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	6	0.376	0.373	0.373	0.374	2.242	2.530
	6	0.433	0.429	0.428	0.430	2.578
	6	0.467	0.473	0.446	0.462	2.770
Test material	1	-0.008	-0.008	-0.008	-0.008	-0.008	0.002
	1	0.003	0.002	0.002	0.002	0.002
	1	0.013	0.014	0.013	0.013	0.013

Calculations are made without rounding off.

¹ OD490 values corrected for the mean final negative control permeability

Table 5

In Vitro Irritancy Score

 

First experiment

 

Treatment	Final Opacity^2	Final OD  ^2 490	In vitro Irritancy Score ^1
Negative control	2.1	0.035	2.6
	2.9	0.015	3.2
	2.5	0.014	2.7
Positive control	96	2.619	135
	131	1.453	153
	108	0.926	122
Test material	2.0	-0.002	1.9
	1.6	-0.003	1.6
	5.5	0.001	5.5

¹ In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

² Positive control and test material are corrected for the negative control.

 

Second experiment

 

Treatment	Final Opacity^2	Final OD  ^2 490	In vitro Irritancy Score ^1
Negative control	2.6	0.011	2.8
	-1.0	0.017	-0.7
	0.3	0.009	0.4
Positive control	103	2.242	136
	101	2.578	139
	67	2.770	109
Test material	6.9	-0.008	6.8
	3.1	0.002	3.1
	-0.4	0.013	-0.2

¹ In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

² Positive control and test material are corrected for the negative control.

 

Table 6

Historical Control Data for the BCOP Studies

 

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Min	-2.42	-0.009	-2.34	69
Max	5.80	0.202	5.90	280
Mean	1.04	0.011	1.21	144
SD	1.63	0.019	1.64	32
n	202	202	202	209

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2019 to May 2022

Interpretation of results:

other: Inconclusive

Conclusions:

Since 3 out of 6 corneas were spread over 2 categories (IVIS <3 and > 3 ≤ 55), the test is inconclusive.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Red HF2 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 potential of the test material 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 SR164:1 of the test material was a dark red solid. Since no workable suspension in physiological saline could be obtained, the test material was used as delivered and added 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 for negative controls indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score (IVIS) of the positive control (20% (w/v) Imidazole) was 137 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.

The test material did not induce ocular irritation through both endpoints (change in opacity or change in permeability), resulting in a mean IVIS of 3.0 after 240 minutes of treatment. Since the result for one of the individual corneas was discordant with the mean IVIS of 3.0 (IVIS of 1.6, 1.9, and 5.5, respectively), and that the discordant IVIS was >10 IVIS units from 55, the test was considered to be inconclusive, and a repeat experiment was performed.

In the second experiment the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range for negative controls indicating that the negative control did not induce irritancy on the corneas. The mean IVIS of the positive control (20% (w/v) Imidazole) was 128 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

The test material did not induce any increase in permeability, and did not increase opacity sufficient for classification, resulting in a mean in vitro irritancy score of 3.2 after

240 minutes of treatment. The results for one of the individual corneas was discordant with the mean IVIS of 3.2 (IVIS of -0.2, 3.1, and 6.8, respectively), and the discordant IVIS was

>10 IVIS units from 55, the test was considered to be inconclusive. A third experiment was considered unnecessary.

Since 3 out of 6 corneas had IVIS values >3 and 3 had IVIS values <3, and the overall mean IVIS was 3.1 (IVIS <3 and > 3 ≤ 55), the test is considered to be inconclusive. However, it should be noted that the OECD 437 test guideline definition of an inconclusive result includes an incoherent ‘AND’ clause for experiments with discordant results at the threshold between IVIS scores of 3 and >3.