[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:

13 - 17 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted in 2020

Deviations:

yes

Remarks:

demonstration of technical proficiency not provided

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

Cas Number:

18641-58-2

Molecular formula:

C69H132O8

IUPAC Name:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Vitelco,’s Hertogenbosch, The Netherlands
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in physiological saline in a suitable container under cool conditions.
- Time interval prior to initiating testing: The eyes were collected from the slaughter house and tested the day of arrival in the laboratory
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were examined 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.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL:
- Amount applied: 750 µL

POSITIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 20% w/v

Duration of treatment / exposure:

240 ± 10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

triplicates for each treatment and control groups

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were excised by a slaughterhouse employee as soon as possible after slaughter. The isolated corneas were stored in a petri dish containing Eagle's Minimum Essential Medium (cMEM) supplemented with 1% v/v L-glutamine and 1% fetal bovine serum. A specifically designed corneal holder was used to mount each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
After an equilibration period, the initial opacity was determined using an opacitometer (BASF-OP3.0, Duratec GmbH, Hockenheim, Germany). Only corneas that had an initial opacity reading higher than 7 were used for the assay.

TREATMENT METHOD: Closed chamber method
The corneas were mounted in a corneal holder with the endothelial side against the O-ring of the posterior half of the chamber. The anterior chamber was then 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 h at 32 ± 1°C.
Following equlibration and an initial opacity reading, the test item, negative or positive control were directly applied on the corneas.
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 min at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions and the test item were removed and the epithelium was washed at least three times with MEM containing phenol red. Possible pH effects of the test item 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.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF-OP3.0, Duratec GmbH). The light was measured as illuminance (I = luminous flux per area, unit: lux).
- Corneal permeability: Sodium fluorescein was distributed over the cornea and the corneas were incubated at 90 ± 5 min at 32 ± 1 °C. The passage of sodium fluorescein dye was measured with the aid of a spectrophotometer (Tecan Infinite M200 Pro Plate Reader) at 490 nm (OD 490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
Test substance with an IVIS > 55 was considered as severe irritant/corrosive and labelled Category 1 according to CLP/EPA/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneae

Run / experiment:

240 ± 10 min exposure

Value:

-2.7

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control responses resulted in opacity and permeability values that were less than the upper limits of the laboratory historical range.
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS value which fell within two standard deviations of the current historical mean.

Any other information on results incl. tables

Table 1: Summary of opacity, permeability and in vitro irritancy scores

	Mean	Mean	Mean In vitro
Treatment
	Opacity	Permeability	Irritation Score*#
Negative control	3.0	0.006	3.1
Positive control	151	1.594	175
Test item	-3.7	0.070	-2.7

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

Table 2: Individual opacity scores

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^§	Negative control corrected Final Opacity^$	Mean Final Opacity
Negative control	2.1	7.6	5.5		3.0
	2.4	5.3	2.8
	2.4	3.0	0.6
Positive control	4.2	157.2	153.0	150	151
	3.9	150.3	146.4	143
	4.9	166.6	161.7	159
	3.4	2.7	-0.8	-3.7
Test item	3.8	2.3	-1.5	-4.5	-3.7
	3.4	3.4	0.0	-3.0

Calculations are made without rounding off. ^§ Final Opacity = Opacity after treatment - Opacity before treatment ^$ Negative control corrected Final Opacity = Final opacity - Mean final opacity negative control

Table 3: Individual permeability scores, uncorrected

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	0.008	0.009	0.009	0.009	0.009	0.006
	1	0.012	0.016	0.015	0.014	0.014
	1	-0.005	-0.003	-0.005	-0.004	-0.004
Positive control	1	1.477	1.482	1.486	1.482	1.482
	6	0.279	0.277	0.276	0.277	1.664
	6	0.212	0.242	0.404	0.286	1.716
	1	0.180	0.178	0.178	0.179	0.179
Test item	1	0.015	0.011	0.008	0.011	0.011
	1	0.038	0.038	0.037	0.038	0.038

Calculations are made without rounding off.

Table 4: Individual permeability scores, corrected

Treatment	Dilution factor	Negative control corrected OD490 1#	Negative control corrected OD490 2#	Negative control corrected OD490 3#	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	1	1.471	1.476	1.480	1.475	1.475	1.594
	6	0.273	0.271	0.270	0.271	1.627
	6	0.206	0.236	0.398	0.280	1.679
	1	0.174	0.172	0.172	0.172	0.172
Test item	1	0.009	0.005	0.002	0.005	0.005	0.070
	1	0.032	0.032	0.031	0.031	0.031

Calculations are made without rounding off. ^#OD490 values corrected for the mean final negative control permeability (0.006).

Table 5: In Vitro Irritancy Score

Treatment	Final Opacity^#	Final OD490^#	In vitro Irritancy Score*
	5.5	0.009	5.6
Negative control	2.8	0.014	3.1
	0.6	-0.004	0.5
	150	1.475	172
Positive control	143	1.627	168
	159	1.679	184
	-3.7	0.172	-1.1
Test item	-4.5	0.005	-4.4
	-3.0	0.031	-2.5

* In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value). # Positive control and test item are corrected for the negative control.

Table 6: Historical control data

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.60 - 6.20	-0.011 - 0.081	-2.70 - 6.30	86 - 251
Mean	1.16	0.012	1.35	150
SD	1.78	0.012	1.83	28
n	180	180	180	183

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 Nov 2017 to Nov 2020.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) 1272/2008.

Conclusions:

The irritation potential of the test substance was assessed in the BCOP assay. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of -2.7. All validity criteria were within acceptable limits. Thus, the test substance is considered to be non-corrosive.