trans-cinnamic acid

Endpoint summary

Description of key information

Key studies are available for skin and eye irritation. These studies are performed in accordance with the appropriate OECD guidelines and under the conditions of GLP.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 23 May 2018 and 25 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

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


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

Test system:

human skin model

Source species:

other: reconstructed human epidermis

Cell type:

other: normal, human-derived epidermal keratinocytes (NHEK)

Cell source:

other: reconstructed human epidermis

Source strain:

other: reconstructed human epidermis

Details on animal used as source of test system:

Not applicable

Justification for test system used:

This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 22 May 2018
EpiDermTM Tissues (0.63cm2) lot number : 28615
Assay Medium lot number : 051718TMA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
2 mL of MTT extractant (isopropanol) was used
to completely immerse each insert and the plate was covered with plate sealer to prevent
Isopropanol evaporation.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Inspected by visual observation
Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
24-well plates; one run

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD570 of test item/mean OD570 of negative control) x 100

Classification of corrosivity potential is based on the relative viabilities for both exposure times according Table 1 below.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

25 mg of test item

Duration of treatment / exposure:

60 minutes/3 minutes

Duration of post-treatment incubation (if applicable):

Overnight

Number of replicates:

24-well plates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean 3 minute exposure period

Value:

96.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean 60 minute

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Direct MTT Reduction

The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

Assessment of Colour Interference with the MTT endpoint

The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Test Item, Positive Control Item and Negative Control Item

Mean OD570 values and viabilities for the negative control, positive control and test item are given in the below table:

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.145	2.217	0.101	4.6	100*
		2.288
	60 Minutes	1.871	1.922	0.071	3.7
		1.972
Positive Control	3 Minutes	0.055	0.051	0.006	n/a	2.3
		0.047
	60 Minutes	0.030	0.040	0.014	n/a	2.1
		0.050
Test Item	3 Minutes	2.115	2.132	0.023	1.1	96.1
		2.148
	60 Minutes	1.766	1.806	0.057	3.1	94.1
		1.846

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minutes	100*	2.3	96.1
60 minutes	100*	2.1	94.0

*The mean viability of the negative control tissues is set at 100%

Quality Criteria

The mean OD570 for the negative control treated tissues was 2.217 for the 3-Minute exposure period and 1.922 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 2.1% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the

corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for

MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minutes	100*	2.3	96.1
60 minutes	100*	2.1	94.0

* The mean viability of the negative control tissues is set at 100%

Quality Criteria

The quality criteria required for acceptance of the results in the test were satisfied

Conclusion

The test item was considered to be non-corrosive to the skin

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study is conducted between 30 May and 04 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

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 termperature, in the dark, over silica gel

Test system:

human skin model

Remarks:

EpiSkin Reconstructed Human Epidermis Model

Source species:

other: not applicable: reconstituted human epidermis model

Cell type:

other: not applicable: reconstituted human epidermis model

Cell source:

other: not applicable: reconstituted human epidermis model

Source strain:

other: not applicable: reconstituted human epidermis model

Details on animal used as source of test system:

not applicable: reconstituted human epidermis model

Justification for test system used:

The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute
consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the
main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin
irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between
Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 18-EKIN-022
- Date Received: 29 May 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by
filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/L
- Incubation time: 42 hour
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: no reference filter
- Filter bandwidth: n/a
- Linear OD range of spectrophotometer: not recorded
- A visual assessment was made

NUMBER OF REPLICATE TISSUES: triplicate tissues


PREDICTION MODEL / DECISION CRITERIA

- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 10mg
- Concentration (if solution): n/a

VEHICLE
- N/A, test item used as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): N/A, used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): 5% w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

81.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a white color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD570 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in the below table. The mean viabilities and standard deviations of the test item and positive control, relative to the

negative control are also given in the below table.

The relative mean viability of the test item treated tissues was 81.9% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.

It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.

Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item	OD570	Mean OD570of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.935	0.879	0.120	106.4	100*	11.6
	0.762			86.7
	0.941			107.1
Positive Control Item	0.062	0.035	0.023	7.1	4.0	2.7
	0.022			2.5
	0.022			2.5
Test Item	0.675	0.720	0.059	76.8	81.9	6.7
	0.787			89.5
	0.699			79.5

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 4.0% relative to the negative control treated tissues and the standard deviation value of the viability was 2.7%.

The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.879 and the standard deviation value of the viability was 11.6%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.7%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was

based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt

(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The

maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified

isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 81.9% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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

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

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.

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

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.

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

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor / 20171106
- Expiration date of the lot/batch: 10 April 2020
- Purity test date: 09/04/2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: stable


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Final preparation of a solid: applied as such

Species:

chicken

Strain:

other: Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Baileys Turkeys Ltd., Cheshire, UK
- Characteristics of donor animals (e.g. age, sex, weight): chickens weighed approximately 3 kg and were approximately 56 days old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- indication of any existing defects or lesions in ocular tissue samples: Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.

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): 0.03 g

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of
the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5.
Acceptable eyes were dissected from the skull and pulled from the orbit by holding the
nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully
removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.
Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure.
Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea.
Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.
After the approval process the eyes were incubated for approximately 45 minutes for
equilibrium purposes.

EQUILIBRATION AND BASELINE RECORDINGS
Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES
The test item and positive control item groups consisted of three eyes and the negative
control item group consisted of two eyes.

NEGATIVE CONTROL USED

Identification: Sodium chloride 0.9% w/v
Batch: 18/10BB1B
Purity: 0.9%
Physical state / Appearance: Clear colorless liquid
Expiry Date: 01 August 2021
Storage Conditions: Room temperature


POSITIVE CONTROL USED
Identification: Imidazole
Batch: 162411
Purity: >99%
Physical state / Appearance: White crystalline powder
Expiry Date: 10 April 2019
Storage Conditions: Room temperature

APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish. 0.03 g of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.
0.03 g of the positive control item, Imidazole, was similarly applied to the cornea of each
positive control eye and 0.03 mL of the negative control item was applied to the cornea of
each negative control eye.

OBSERVATION PERIOD
Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes
(±5 minutes) after the eyes had been decontaminated with the isotonic saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL isotonic saline

METHODS FOR MEASURED ENDPOINTS:
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein
retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium).
All of the endpoints, with the exception of fluorescein retention (which is only determined at
30 minutes after test substance exposure) were determined at each of the above time points.
- Corneal opacity: Percentage corneal swelling was assessed from corneal thickness measurements. Mean percentage of corneal swelling for all test eyes was calculated for all the time points.
The overall category score was determined from the highest mean score for epithelial
swelling as observed at any time point.
Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
- Damage to epithelium based on fluorescein retention:
The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time
interval only. These measurements are used for the overall classification for each test item.
- Macroscopic morphological damage to the surface:
Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all
morphological effects that can be noted on the cornea. The classification of these findings
was subject to interpretation.


SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Irritation parameter:

cornea opacity score

Run / experiment:

maximal mean

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean fluorescien retention

Value:

0.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Irritation parameter:

percent corneal swelling

Run / experiment:

Maximal mean

Value:

12.27

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: ‘Easily discernible translucent area; details of the iris are clearly visible’ of opacity were noted in the test item treated eyes.
No morphological effects were noted in the test item, positive or negative control item treated eyes.
Very minor single cell staining was noted in two test item treated eyes. Single cell staining scattered throughout the treated area of the cornea was noted in one test item treated eye.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:

Maximal mean score for corneal opacity : 0.5 ICE Class I
Mean score of Fluorescein Retention : 0.3 ICE Class I
Maximal mean corneal swelling compared to time zero : 0.00% ICE Class I
Very faint opacity was noted in the negative control treated eyes.
No fluorescein retention was noted in one negative control treated eye during the study period. Very minor single cell staining was noted in one negative control treated eye.

- Acceptance criteria met for positive control:

Maximal mean score for corneal opacity : 4.0 ICE Class IV
Mean score of Fluorescein Retention : 3.0 ICE Class IV
Maximal mean corneal swelling compared to time zero : 24.55% ICE Class III
‘Complete corneal opacity; iris invisible’ was noted in all positive control treated eyes.
‘Confluent large areas of the cornea retaining fluorescein’ was noted in all positive control treated eyes.

- Range of historical values if different from the ones specified in the test guideline:

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made following assessment of the data for all endpoints.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

Skin irritation: The substance is not considered to be classified in accordance with Regulation (EC) No. 1272/2008.

Eye irritation: The results of the in vitro/ex-vivo studies were inconclusive and as such a classification of Category 2 eye irritant is proposed on the basis of the following justification and bearing in mind the animal welfare aspects of in vivo testing:

Neither the result of the OECD 437 BCOP test or the OECD 438 BCOP test indicates potential for serious eye damage and whilst these tests have reported false negative rates for prediction of ‘Category 1’ of 14% & 48% respectively, it is unlikely that the substance is corrosive to the eyes. It can be reasonable anticipated that the substance might be irritating however, and as such, a classification of Category 2 is proposed.

No further testing is proposed since for EU REACH, and for animal welfare reasons, testing in animals should be conducted only as a last resort.