[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:

weight of evidence

Study period:

July 4, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

BCOP test was performed to identify test substance that can induce serious eye damage and to identify test substance not requiring classification for eye irritation or serious eye damage.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

yes

Remarks:

Deviation was considered to have not affected the integrity or validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C16-18 and ethoxylated C16-18 , phosphates (20 moles ethoxylation)

Molecular formula:

C18H39O4P1 (monoester representative, i.e., mono- C18 PSE) C56H115O24P1 (ethoxylated monoester representative, i.e., mono- C16 AE20 PSE) C32H67O4P1 (diester representative, i.e., di- C18 PSE) C112H227O44P1 (ethoxylated diester representative, i.e., di- C16 AE20 PSE)

IUPAC Name:

Alcohols, C16-18 and ethoxylated C16-18 , phosphates (20 moles ethoxylation)

Test material form:

solid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20% w/v test substance in sodium chloride 0.9% w/v

Duration of treatment / exposure:

240 minutes

Details on study design:

Preparation of corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of corneas and opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.

Treatment of corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance preparation or control substances were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 240 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. The negative and positive control data was shared with Envigo - Shardlow study number NX25RF and XL29CC.

Application of sodium fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes.

Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Data evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test substance induced a response through only one of the two endpoints.

Visual observation
The condition of the cornea was visually assessed post treatment.

Major Computerized Systems
The following computerized systems were used in the study:
• Delta Building Monitoring System.
• Labtech LT-4500 microplate reader and LT-com software

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 51.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction could be made about eye irritation potential

Any other information on results incl. tables

Results

Individual and mean corneal opacity and permeability measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control*	1	3	4	1		0.012
	2	3	4	1		0.000
	3	2	4	2		0.000
	Mean			1.3		0.004		1.4
Positive Control*	4	2	105	103	101.7	3.965	3.961
	5	2	88	86	84.7	1.895	1.891
	6	2	83	81	79.7	1.820	1.816
	Mean				88.7		2.556	127.0
Test Substance	13	3	74	71	69.7	0.008	0.004
	14	2	38	36	34.7	0.011	0.007
	15	3	53	50	48.7	0.000	0.000
	Mean				51.0		0.004	51.1

OD= Optical density            

* = Control data shared with Envigo - Shardlow study number NX25RF and XL29CC

Corneal epithelium condition post treatment

Treatment Cornea Number Observation Post Treatment Negative Control* 1 Clear 2 Clear 3 Clear Positive Control* 4 Cloudy 5 Cloudy 6 Cloudy Test Substance 13 Cloudy 14 Cloudy 15 Cloudy

*= Control data shared with Envigo - Shardlow study number NX25RF and XL29CC

 

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

 

Treatment	In Vitro Irritancy Score
Test substance	51.1
Negative Control	1.4
Positive Control	127.0

 

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was above the range of 65.1 to 123.3. The positive control acceptance criterion was therefore not satisfied. This was reported as a deviation. The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

Deviation

The positive control group had an overall IVIS of 127.0. This was marginally higher than the criteria range set for an acceptable test. However, as the score was only marginally exceeded, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant. This deviation was considered to have not affected the integrity or validity of the study.

Applicant's summary and conclusion

Interpretation of results:

other: CLP - inconclusive results

Conclusions:

Under the study conditions, no prediction of eye irritation for the test substance could be made.

Executive summary:

A study was performed to determine the eye irritation potential of test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', using BCOP test, according to EU Method B.47 and OECD Guideline 437, in compliance with GLP. Appropriately prepared bovine cornea, in triplicates were expsured to 0.75 mL of the test substancs (20% w/v in sodium chloride 0.9% w/v ) and reference substances (Negative control: sodium chloride 0.9% w/v, Positive control: Imidazole 20% w/v solution in sodium chloride 0.9% w/v). The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 240 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’s Minimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The negative and positive control data was shared with Envigo - Shardlow study number NX25RF and XL29CC. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS for test substance, negative control and positive control were reported to be 51.1, 1.4 and 127 respectively. The study has met all validity criteria, except for positive control, however, the deviation was considered to have not affected the integrity or validity of the study. Under the study conditions, based on the IVIS score of the test substance, which is >3 and <55%, no prediction of eye irritation potential could be made (Envigo, 2017).