Reaction mass of 2-[[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]methyl]oxirane and 1,3-bis[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol and 1-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-3-[4-[[4-[3-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-2-hydroxy-propoxy]-3,5-dimethyl-phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

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:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]propan-2-ol; 1-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-3-{4-[(4-{3-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-2-hydroxypropoxy}-3,5-dimethylphenyl)methyl]-2,6-dimethylphenoxy}propan-2-ol; 2-{[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]methyl}oxirane

EC Number:

941-357-0

Molecular formula:

Not applicable for multi-constituent substances

IUPAC Name:

1,3-bis[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]propan-2-ol; 1-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-3-{4-[(4-{3-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-2-hydroxypropoxy}-3,5-dimethylphenyl)methyl]-2,6-dimethylphenoxy}propan-2-ol; 2-{[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]methyl}oxirane

Test material form:

other: Liquid

Details on test material:

Batch: DG4K30997A
Test substance storage: At room temperature protected from light
Stable under storage conditions until 25 October 2015 (expiry date)

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Preparation of corneas:
All eyes were carefully examined for defects by holding the eyes to the light after submerging in physiological saline. Those exhibiting unacceptable
defects, such as opacity, scratches, pigmentation and neovascularization were discarded.

The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands)
containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)).
The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, 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.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

Test material applied in excess

Duration of treatment / exposure:

120 ± 10 minutes at 32 ± 1°C

Number of animals or in vitro replicates:

Three eyes for the test substance, negative control and positive control substance.

Details on study design:

Details on treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 ul of the negative control and 10% (w/v) Benzalkonium chloride solution (positive control) were introduced onto the epithelium of the cornea. The test substance was applied directly on the corneas in such a way that the cornea was completely covered (in excess).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 10 ± 1 minutes at 32 ± 1°C, and then subsequently washed and possible pH effects noted. Subsequently the corneas were incubated for a further 240 ± 10 minutes at 32 ± 1°C. After the
incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). After the completion of the incubation period each cornea were inspected visually for dissimilaropacity patterns and the opacity determination was performed.

Opacity measurements:
The opacitometer determined the difference in the light transmission between each control or test substance treated cornea and an air filled chamber.The numerical opacity value (arbitrary unit) was displayed and recorded. 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 positive
control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the changein opacity of each positive control or test substance 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.

Permeability determination:
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 ul 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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before thedilution factor was applied to the readings.

Results and discussion

In vitro

Results

Irritation parameter:

cornea opacity score

Value:

>= -2 - <= -1

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls were 0.0 for all three corneas. The individual positive control in vitro irritancy scores ranged from 132 to 165. The corneas treated with the positive control were turbid after 10 minutes of treatment.

The corneas treated with the test substance showed opacity values ranging from -2.0 to -1.0 and permeability values ranging from -0.002 to 0.006. The corneas were clear after the 10 minutes of treatment. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.0 to -0.9 after 10 minutes of treatment.

Any other information on results incl. tables

Treatment	Mean Opacity	Mean Permeability	Mean In vitro irritation Score
Negative control	0.0	0.0	0.0
Positive control (Benzalkonium Chloride)	103.0	3.287	152.3
Test substance	-1.3	0.002	-1.3

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

Since the test substance induced an in vitro irritancy score (IVIS) ≤ 3, no classification is required for eye irritation or serious eye damage