[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:

16-07-2019 to 15-11-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

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)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

{2-[(2-methylundec-1-en-1-yl)oxy]ethyl}benzene

Cas Number:

2489743-82-8

Molecular formula:

C20H32O

IUPAC Name:

{2-[(2-methylundec-1-en-1-yl)oxy]ethyl}benzene

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light container flushed with nitrogen
- Other: colourless liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): > 9 months old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. Post-excision, the isolated eyes were stored in physiological saline in the cooled slaughter-house until transportation to the laboratory using a suitable container. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: < 24 hours. Corneas were prepared for testing immediately on same day arrival.
- indication of any existing defects or lesions in ocular tissue samples: None. Only corneas free from damage utilised (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches were discarded).
- Indication of any antibiotics used: None reported.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

10 minutes at 32 ± 1ºC.

Duration of post- treatment incubation (in vitro):

The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

Number of animals or in vitro replicates:

Three (3) per test item, or negative or positive controls, respectively.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) 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. 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. The opacity of each cornea was read against an air 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.
SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder, details on cornea holder in the full study report) 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. 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 (details in the full study report). The opacity of each cornea was read against an air 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.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches) and where necessary discarded. Additionally, only corneas with opacity < 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: physiological saline solution (recognised supplier ; documented in the full study report)

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: neat Ethanol (recognised supplier ; documented in the full study report)

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 ± 1 minutes at 32 ± 1ºC.

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium) 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.
- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: Opacity, Permeability and In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (n=3)

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

IVIS scores ranged from 1.7 to 3.2 (n=3) with mean 2.4 after 10 minutes treatment ; individual scores = 2.3, 3.2 and 1.7, respectively

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None. No pH effect of the test item was observed on the rinsing medium.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. 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. One of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base. This deviation had no impact on the study since two negative control eyes were available and yielded results within the historical control range.
- Acceptance criteria met for positive control: Yes. The mean in vitro irritancy score of the positive control neat ethanol was 55 and was within the historical positive control data range (HCD range : 24.0 to 89.6 and mean = 51.31 ; n=108).
- Range of historical values if different from the ones specified in the test guideline: The mean in vitro irritancy score of the negative control were less than the upper limits of the laboratory historical range and for the positive control neat ethanol at 55..0 and was within the historical positive control data range. 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 2.4 after 10 minutes of treatment. Applicant assessment of the data indicates that the positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively. It was noted one of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base. This deviation had no impact on the study since two negative control eyes were available and yielded results within the historical control range..

Any other information on results incl. tables

Table 1.0 - Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score #1, #2
Negative control	1.6	-0.040	1.6
Positive control	24	2.060	55.0
Test item	2.3	0.005	2.4

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

#2 Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

 

Table 2.0 - Opacity score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity #1	Negative control corrected Final Opacity #2	Mean Final Opacity
Negative control	3.6	6.3	2.7		1.6
	4.7	4.3	0.5
	4.6 #3	9.8 #3	5.2 #3
Positive control	2.7	25.4	22.8	21	24
	4.3	28.2	23.9	22
	4.1	35.3	31.2	30
Test item	4.3	8.2	3.9	2.3	2.3
	3.7	8.4	4.7	3.1
	2.3	5.5	3.2	1.6

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

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

#3 Excluded from analysis (above historic control range)

#4 Calculations are made without rounding off

 

Table 3.0 - Permeability score individual values (uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD		Mean final negative control
Negative control	1	-0.005	-0.005	-0.005	-0.005		-0.005	-0.040
	1	-0.004	-0.001	-0.004	-0.003		-0.003
	1	-0.0028 #2	0.030 #2	0.029 #2	0.029 #2		0.029 #2
Positive control	6	0.368	0.365	0.364	0.366		2.194
	6	0.423	0.430	0.431	0.428		2.568
	1	1.369	1.361	1.368	1.366		1.366
Test item	1	-0.004	-0.004	-0.004	-0.004		-0.004
	1	0.002	0.004	0.008	0.005		0.005
	1	0.005	0.001	0.001	0.002		0.002

#1 Calculations are made without rounding off.

#2 Excluded from analysis (above historic control range)

 

Table 4.0 - In vitro irritancy score

Treatment	Final Opacity #1	Final OD4902	In vitro Irritancy Score #2
Negative control	2.7	-0.005	2.6
	0.5	-0.003	0.5
	5.2 #3	0.029 #3	5.7 #3
Positive control	21	2.218	54
	22	2.592	61
	30	1.370	50
Test item	2.3	0.000	2.3
	3.1	0.009	3.2
	1.6	0.006	1.7

#1 Positive control and test item are corrected for the negative control.

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

#3 Excluded from analysis (above historic control range)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not considered to be irritant in the in vitro Bovine Corneal Opacity and Permeability model. The in mean vitro irritancy score (IVIS) was < 3.0 in the prediction model.

Executive summary:

The study was performed according to OECD TG 437 to assess the eye irritancy potential of the item in isolated bovine corneas in accordance with GLP. The purpose of this test was to evaluate the test item for its potential to induce eye damage/ocular irritation. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. A total of three corneas per treatment group were used. A volume of 750 microlitres of the item was placed the cornea. The negative control group received physiological saline and the positive control group received neat Ethanol. For each group the corneas were incubated for 10 ± 1minutesat 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Eagle’s Minimum Essential Medium) 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. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. 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. One of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base. This deviation had no impact on the study since two negative control eyes were available and yielded results within the historical control range. The mean in vitro irritancy score of the positive control (neat ethanol) was 55.0 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment. Under the conditions of this study, the item is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.