[No public or meaningful name is available]

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test 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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2,3; 2,4; 2,5 and 2,6 mixed xylidenes, C7 and C8 linear substituted naphthols and diazonium salts

Molecular formula:

Not available: variable

IUPAC Name:

Reaction products of 2,3; 2,4; 2,5 and 2,6 mixed xylidenes, C7 and C8 linear substituted naphthols and diazonium salts

Test material form:

solid: particulate/powder

Details on test material:

Batch No. SR164:1
Appearance Dark red solid (see certificate of analysis)
Test substance storage Ambient conditions but protected from temperatures below 5°C

Stability under storage Stable under recommended storage conditions.
Manufacturing Date 12 July 2022
Expiry date 12 July 2027

Specific details on test material used for the study:

No further details in the study report

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.