trans-cinnamic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted on 19 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

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 (incl. QA statement)

Remarks:

Date of Inspection: 18/07/2017 - 20/07/2017 Date of Issue: 28/11/2017

Test material

Test material information

Constituent 1

Reference substance name:

trans-cinnamic acid

EC Number:

205-398-1

EC Name:

trans-cinnamic acid

Cas Number:

140-10-3

Molecular formula:

C9H8O2

IUPAC Name:

3-phenylacrylic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20171106
- Expiration date of the lot/batch: 10 April 2020
- Purity test date: 99.0% w/w

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark, over silica gel

Test animals / tissue source

Species:

other: excised bovine corneas

Strain:

other: excised bovine corneas

Details on test animals or tissues and environmental conditions:

Source of Bovine Eyes
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:

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): 0.1970 g
- Concentration (if solution): N/A

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

N/A

Number of animals or in vitro replicates:

Three

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 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 ± 1 ºC for 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

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 item and three corneas to the positive control item.


NUMBER OF REPLICATES
Three

NEGATIVE CONTROL USED
Identification: Sodium chloride 0.9% w/v
Lot: 3012487
Purity: 0.9%
Supplier: Aguettant Ltd
Expiry Date: 01 November 2018
Storage Conditions: Room temperature

POSITIVE CONTROL USED
Identification: Imidazole
Batch: 162411
Supplier: Fisher Scientific
Purity: >99%
Expiry Date: 10 April 2019
Storage Conditions: Room temperature in the dark

APPLICATION DOSE AND EXPOSURE TIME
0.1970g; 240 minutes

TREATMENT METHOD:
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 0.1970 g of the solid test item was found to adequately cover the corneal surface. 0.75ml of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.


POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red
- POST-EXPOSURE INCUBATION: N/A. 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Using a calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
- Others: pertinent visual observations

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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 item induced a response through only one of the two endpoints

DECISION CRITERIA: In accordance with OECD TG

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

36.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None noted

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - the test is considered acceptable if if the negative control produced an In Vitro Irritancy Score which is less than or equal to theupper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control which in this case was ≤2.3 for opacity and ≤0.044 for permeability. These criteria were satisifed
- Acceptance criteria met for positive control: Yes - the test is considered acceptable if the positive control produced an in vitro irritancy score which fell within two standard deviations of the historical mean during 2017 for this testing facility which in this case was 71.2 to 132.9. The in vitro irritancy score was 81.7 therefore this is satisfied.

Any other information on results incl. tables

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in the table below.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea	Opacity				Permeability (OD492)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment – Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	7	12	5		0.004
	2	5	6	1		0.005
	3	6	6	0		0.003
				2.0*		0.004*		2.1
Positive Control	4	5	67	62	60.0	1.815	1.811
	5	5	74	69	67.0	1.116	1.112
	6	3	64	61	59.0	1.030	1.026
					62.0		1.316*	81.7
Test Item**	7	5	55	50	48.0	0.037	0.033
	10	6	43	37	35.0	0.012	0.008
	11	5	32	27	25.0	0.005	0.001
					36.0*		0.014*	36.2

OD = Optical Density; * = Mean ** = Test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v.

Corneal Epithelium Condition

The condition of each cornea is given in the table below.

Treatment	Cornea Number	Observation Post-Treatment
Negative Control	1	Clear
	2	Clear
	3	Clear
Positive Control	4	Cloudy
	5	Cloudy
	6	Cloudy
Test Item*	7	Cloudy
	10	Cloudy
	11	Cloudy

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.

In Vitro Irritancy Score

The in vitro irritancy scores are summarised as follows.

Treatment	In VitroIrritancy Score
Test Item	36.2
Negative Control	2.1
Positive Control	81.7

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction of eye irritation can be made.

Executive summary:

Introduction

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 cornea in 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.

Method

The test item was applied neat 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).

Data Interpretation

IVIS	UN GHS
≤3	No category
>3;≤55	No prediction can be made
> 55	Category 1

Results

The In Vitro irritancy scores are summarized as follows:

Treatment	In VitroIrritancy Score
Test Item	36.2
Negative Control	2.1
Positive Control	81.7

Conclusion

No prediction of eye irritation can be made.