Ethanone, 1-[4-(acetyloxy)-3-[(acetyloxy)methyl]phenyl]-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2015 - 16 February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed in a GLP lab in accordance with OECD test guidelines with one minor deviation that was considered not to affect the integrity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

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:

yes

Remarks:

Control groups data and documentation were shared with another study. This deviation was considered not to affect the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[5-acetyl-2-(acetyloxy)phenyl]methyl acetate

EC Number:

921-042-4

Cas Number:

24085-06-1

Molecular formula:

C13-H14-O5

IUPAC Name:

[5-acetyl-2-(acetyloxy)phenyl]methyl acetate

Constituent 2

Reference substance name:

4-acetyl-2-[(acetyloxy)methyl]phenyl acetate

IUPAC Name:

4-acetyl-2-[(acetyloxy)methyl]phenyl acetate

Test material form:

other: Off white solid blocks

Details on test material:

- Name of test material (as cited in study report): Diacetate
- Expiration date of the lot/batch: 16 June 2015
- Storage condition of test material: Room temperature in the dark

Test animals / tissue source

Species:

other: Cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Local abattoir
- Age at study initiation: 12 to 60 months old

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 vehicle

Amount / concentration applied:

0.75 mL of the test substance present at 20% w/v in a 0.9% w/v sodium chloride solution.

Duration of treatment / exposure:

240 minutes followied by three rinces in fresh minimum essential medium (MEM).

Observation period (in vivo):

Opacity readings were taken and coreneas were observed visually following MEM rince. Permeability to sodium fluoresein was als evaluated.

Number of animals or in vitro replicates:

Nine eyes were used during the study, corneas with opacity values close to the median value were used.
Three corneas were issued to each group, test group, positive and negative control group.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: Corenas were rinced three times with fresh MEM.
- Time after start of exposure: 240 minutes


SCORING SYSTEM:

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 OD492 value).
In Vitro Irritancy Score: ≤ 3 - No category. Not requiring classification to UN GHS or EU CLP
In Vitro Irritancy Score: > 3; ≤55 - No prediction of eye irritation can be made
In Vitro Irritancy Score > 55 -Category 1. UN GHS or EU CLP Causes serious eye damage

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.


TOOL USED TO ASSESS SCORE: fluorescein permeability

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

Negative Control

Basis:

mean

Time point:

other: 240 minutes

Score:

ca. 2.2

Reversibility:

not specified

Results 2

Irritation parameter:

cornea opacity score

Remarks:

Positive Control

Basis:

mean

Time point:

other: 240 minutes

Score:

ca. 97.7

Reversibility:

not specified

Results 3

Irritation parameter:

cornea opacity score

Remarks:

Test substance

Basis:

mean

Time point:

other: 240 minutes

Score:

ca. 66.1

Reversibility:

not specified

Irritant / corrosive response data:

The In Vitro irritancy scores are summarized as follows:
Test Item: 66.1
Negative Control: 2.2
Positive Control: 97.7

Other effects:

The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment

Any other information on results incl. tables

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number				Opacity												Permeability (OD)			InVitroIrritancyScore
					Pre-Treatment		Post-Treatment			Post-Treatment− Pre-Treatment			Corrected Value						Corrected Value
Negative Control⊕	12				3		6			3							0.009
	18				3		5			2							0.012
	19				3		4			1							0.014
										2.0*							0.012♦			2.2
Positive Control⊕	1				6		85			79			77				1.505		1.493
	3				2		77			75			73				1.393		1.381
	5				4		82			78			76				1.601		1.589
													75.3•						1.488•	97.7
Test Item	14				1		69			68			66				0.042		0.03
	16				1		67			66			64				0.047		0.035
	20				2		71			69			67				0.04		0.028
													65.7•						0.031•	66.1

OD = Optical density

* = Mean of the post-treatment − pre-treatment values

♦ = Mean permeability

• = Mean corrected value

⊕ = Control group shared with Harlan study number 41500024

Table 2: Corneal Epithelium Condition Post Treatment

Treatment

Cornea Number

Observation

Post Treatment

NegativeControl⊕

12

Clear

18

Clear

19

Clear

PositiveControl⊕

1

Cloudy

3

Cloudy

5

Cloudy

Test Item

14

Cloudy

16

Cloudy

20

Cloudy

⊕ = Control group shared with Harlan study number 41500024

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Category 1 Criteria used for interpretation of results: EU

Conclusions:

Category 1. UN GHS or EU CLP Causes serious eye damage.

Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. 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 test item is classified according to the prediction model below:

IVIS	CLASSIFICATION
≤ 3	No category. Not requiring classification toUNGHS or EUCLP
>3; ≤55	No prediction of eye irritation can be made
>55	Category 1.UNGHS or EUCLP Causes serious eye damage

 

The In-Vitro irritancy scores are summarized as follows:

Treatment	InVitroIrritancy Score
Test Item	66.1
Negative Control	2.2
Positive Control	97.7

 

 

The test Item was concluded to be an Eye Irritant, Category 1. UNGHS or EUCLP Causes serious eye damage.