Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation/corrosion:

According to the assay caried out following OECD TG 439, the substance was not found to be irritating.

Eye irritation/corrosion

According to the assay caried out following OECD TG 437, no conclusion could be made on the irritancy potential of the substance.

According to the assay caried out following OECD TG 492, the substance was found to be irritating to eyes category 1 or 2.

Following the weight of evidence below, the substance was classified as EDI Category 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 24 August 2016 Experimental completion date: 29 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
Please see principles of method below for details.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Please see principles of method below for details.
Principles of method if other than guideline:
Deviation 1
During the determination of test item doses required for the direct reduction of MTT, color interference and also tissue dosing the mean quantity was found to be greater than 10 mg.
The actual mean value was found to be 12.4 mg determined over 4 individual measurements.
This was considered acceptable as the doses applied were in excess of 10 mg and therefore on the side of caution.
This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes
Specific details on test material used for the study:
Information as provided by the Sponsor. A certificate of analysis supplied by the sponsor is presented as.

Identification: EVA
Common name: ETHYLVANILLYL ALCOHOL, White Vanilla
CAS Number: 4912-58-7
CAS Name: Benzenemethanol, 3-ethoxy-4-hydroxy-
Chemical Name: 2-Ethoxy-4-(hydroxymethyl)phenol
Physical state/Appearance: Pale yellow powder
Storage Conditions: room temperature in the dark
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not stated
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic Laboratories, Lyon, France
- Tissue batch number(s):
EpiSkinTM Tissues (0.38cm2) lot number : 16-EKIN-034
Maintenance Medium lot number : 16-MAIN3-056
Assay Medium lot number : 16-ESSC-037
- Delivery date: 23 August 2016
- Date of initiation of testing: 24 August 2016

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

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for
15 minutes.

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. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the relative mean tissue viability is less than 50%.
- The test substance is considered to be non-corrosive to skin if the relative mean tissue viability is greater than or equal 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
other: A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Used as supplied.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Prepared as a 5% w/v aqueous solution.
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 viability
Value:
91.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- 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.
- Colour interference with MTT: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.098 and the standard deviation value of the viability was 4.1%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 9.2% relative to the negative control treated tissues and the standard deviation value of the viability was 0.8%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.4%. The test item acceptance criterion was therefore satisfied.

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD562of tissues

Mean OD562of triplicate tissues

+/- SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

+/- SD of Relative mean viability (%)

Negative Control Item

1.099

1.098

0.045

100.1

100*

4.1

1.053

95.9

1.143

104.1

Positive Control Item

0.108

0.101

0.009

9.8

9.2

0.8

0.091

8.3

0.105

9.6

Test Item

1.130

1.008

0.125

102.9

91.8

11.4

0.880

80.1

1.013

92.3

Interpretation of results:
other: EU CLP Not classified for irritation.
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).
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 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 562 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 91.8% 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. The following classification criteria apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 October 2017 to 28 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 492 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
(Adopted 28 July 2015)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, protected from light
Species:
human
Details on test animals or tissues and environmental conditions:
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium.

Assay medium: OCL-200-ASY/Assay Medium
MTT diluent: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ and OCL-200-ASY/Assay Medium used for diluting MTT
Pre-treatment / wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+
Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT), 1.0 mg / mL MTT diluent
Extracting agent: 2-propanol
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): 50 mg

CONTROLS
- Control tissues were concurrently applied with 50 μL of sterile distilled water (NC) or with 50 μL of methyl acetate (PC).
Duration of treatment / exposure:
6 hours ± 15 minutes
Duration of post- treatment incubation (in vitro):
18 hours ± 15 minutes
Number of animals or in vitro replicates:
2
Details on study design:
EXPERIMENTAL PROCEDURE

To assess the ability of the test material to directly reduce MTT a pretest was performed. Fifty milligrams of the test substance was added to 1 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 180 minutes. As a result, the change in color was not observed. However, the test substance was changed color in to light purple and it was judged that the test substance had reactivity with MTT. Therefore, interference of the test substance with MTT (interference test) was conducted in the eye irritation test.
-Basic procedure:
Two tissues were treated with each, the test substance, the PC and the NC.
Pre-incubation of the tissues:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 60 ± 5 minutes the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 - 24 hours.
-Pretreatment of the tissues:
After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
-Application of the test substance:
Fifty milligrams of the test material was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile distilled water (NC) or with 50 μL of methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 6 hours ± 15 minutes was completed.
-Removal of the test substance and postincubation period:
To remove the test substance, the tissues were washed with sterile PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium in order to remove residual test substance. After 25 ± 2 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours ± 15 minutes.
-MTT incubation:
After the post-incubation period, the assay medium was replaced by 0.3 mL/well MTT solution and the tissues were incubated in the incubator for 180 ± 10 minutes.
After incubation, the outside of tissue inserts was wiped. All tissues were transferred into new 6-well plates filled with 2 mL/well of 2-propanol. The plate was put into a plastic bag, and extraction was performed at room temperature for 2 hours or more using a plate shaker. The extracts were mixed to obtain homogeneous solutions.
-Optical Density measurements:
The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 8 microtiter wells filled with 2-propanol in a microtiter plate.
.
- RhCE tissue construct used, including batch number:
Tissue model: OCL-200
Tissue Lot Number: 20994
Supplier: MatTek Corporation

- Evaluation of results:
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile distilled water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60%.
Irritation parameter:
other: relative mean viability of the tissues (%)
Run / experiment:
6 hours
Value:
7.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no
Interpretation of results:
other: Category 1 (irreversible effects on the eye) or Category 2 (irritating to eyes) based on GHS criteria
Remarks:
See section 13.2 for classification justification
Conclusions:
Based on the observed results and applying the evaluation criteria it was concluded, that White vanilla shows a serious eye damage or an eye irritation potential in the EpiOcularTM in vitro eye irritation test under the test conditions chosen.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 01 September 2016 Experimental completion date: 01 September 2016
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
Specific details on test material used for the study:
Information as provided by the Sponsor. A Certificate of Analysis provided by the sponsor is presented as.

Identification: EVA
Common name: ETHYLVANILLYL ALCOHOL, White Vanilla
CAS Number: 4912-58-7
CAS Name: Benzenemethanol, 3-ethoxy-4-hydroxy-
Chemical Name: 2-Ethoxy-4-(hydroxymethyl)phenol
Storage Conditions: room temperature in the dark
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): Eyes were obtained from adult cattle (typically 12 to 60 months old).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics. They were transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: The corneas were prepared immediately on arrival.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: Prior to transportation the eyes were were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

Vehicle:
physiological saline
Remarks:
0.9% w/v sodium chloride solution.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% w/v solution in 0.9% w/v sodium chloride solution.

VEHICLE
- Amount(s) applied (volume or weight with unit): as above
- Concentration (if solution): Sodium chloride 0.9% w/v
- Lot/batch no. (if required): 3011424
- Purity: 0.9%
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
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 60 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. The average opacity for all corneas was calculated.

NUMBER OF REPLICATES
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

NEGATIVE CONTROL USED
The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.

SOLVENT CONTROL USED (if applicable)
For the purpose of this study the test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.

POSITIVE CONTROL USED
The positive control item, Imidazole, was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.

APPLICATION DOSE AND EXPOSURE TIME
Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were 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.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. 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.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The average opacity for all corneas was calculated using a calibrated opacitometer.
- Corneal permeability: The optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.
- Others: Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
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 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.
Visual Observation
The condition of the cornea was visually assessed post treatment.

DECISION CRITERIA: The test item was classified according to the prediction model in the table below.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean In Vitro Irritancy Score
Value:
4.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: The corneas treated with the test item were clear 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in the following table:

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment – Pre-Treatment

Corrected Value

 

Corrected Value

Negative Control

11

4

6

2

 

0.018

 

 

14

4

7

3

 

0.026

 

 

17

4

5

1

 

0.011

 

 

 

 

 

2.0*

 

0.018♦

 

2.3

Positive Control

10

1

74

73

71.0

1.196

1.178

 

12

3

77

74

72.0

1.840

1.822

 

15

7

75

68

66.0

1.162

1.144

 

 

 

 

 

69.7•

 

1.381•

90.4

Test Item

13

3

12

9

7.0

0.060

0.042

 

16

5

8

3

1.0

0.023

0.005

 

18

2

9

7

5.0

0.058

0.040

 

 

 

 

 

4.3•

 

0.029•

4.8

OD = Optical density       * = Mean of the post-treatment − pre-treatment values       ♦ = Mean permeability       • = Mean corrected value 

Corneal Epithelium Condition

The condition of each cornea is given in the following table:

Treatment

Cornea Number

Observation Post Treatment

Negative Control

11

Clear

14

Clear

17

Clear

Positive Control

10

Cloudy

12

Cloudy

15

Cloudy

Test Item

13

Clear

16

Clear

18

Clear

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

4.8

Negative Control

2.3

Positive Control

90.4

Interpretation of results:
other: No prediction of eye irritation can be made.
Remarks:
See section 13.2 for classification justification
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 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).

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS

Classification

<3

No category. Not requiring classification to UN GHS or EU CLP

> 3; <55

No prediction of eye irritation can be made

> 55

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

Results

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

4.8

Negative Control

2.3

Positive Control

90.4

Conclusion

No prediction of eye irritation can be made.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Justification for classification or non-classification

Skin irritation/corrosion:

Not irritating

Eye irritation/corrosion

To classify the substance Ethyl Vanillyl Alcohol (EC 674-192-5 / CAS 4912-58-7) for Eye irritation/corrosion, in vitro assays have been carried out as follows:

- Test No. 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

The assay was performed in compliance with GLP standards. Furthermore, the criterias for an acceptable test were satisfied and the test was judged acceptable. The in vitro irritation score (IVIS) was found to be 4.8. The IVIS is comprise between 3 and 55 so “no prediction of eye irritation can be made”, according to the Decision Criteria table stated in the test guideline document. Therefore, the result of this assay is not sufficient for classification.

- Test No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage

The assay was conducted in compliance with GLP principles. The criterias for an acceptable test were satisfied and there were no factors which affected the reliability of the test. As a result of the assay above, the cell viability was found to be 7.5%, which was below the 60% judgement criteria. Therefore, the substance was classified as Irritant (UN GHS Category 1 or 2).

In order to avoid an in vivo assay due to animal welfare considerations, the test substance Ethyl Vanillyl Alcohol will be classified according to the two in vitro tests previously described following a weight of evidence approach.

Based on the GHS regulation (United Nations, Globally Harmonized System of classification and labelling of chemicals (GHS), 7th Revised Edition of the GHS "Purple Book", 2017, 534 p.), several steps from figure 3.3.1, page 142 have been followed for classification.

No existing human or animal serious eye damage/ eye irritation are available, and the skin corrosion data suggests that the substance is not classified for skin irritation/corrosion. Also, there is no pH extreme and the structure is not in the QSAR Toolbox, so it was not possible to generate any SAR comparisons.

This leads to Step 6, which is consideration of the total weight of evidence.

o In the mouse Local Lymph Node Assay, no skin irritation was observed up to the highest dose tested, which was 20% (as measured by visual scoring for erythema, and ear thickness). This supports the conclusion that White Vanilla would not be severely irritating or corrosive to the eyes, and so the Category 1 could be excluded.

o An in vitro skin irritation test was conducted with Ethyl Vanillyl Alcohol using the EpiSkin Reconstituted Human Epidermis Model. When tested undiluted, Ethyl Vanillyl Alcohol was classified as a non-irritant, according to the criteria in the protocol. As a result, Ethyl Vanillyl Alcohol would not be classified for irritation under EU CLP and UN GHS. Since Ethyl Vanillyl Alcohol was non-irritating at 100% in an in vitro human skin model, this again supports the conclusion that if Ethyl Vanillyl Alcohol has any effect on the eyes, it is likely to be minor (irritating and not severely irritating or corrosive).

o Vanillyl Butyl Ether (VBE) (EC 444-010-7) has some structural similarity to Ethyl Vanillyl Alcohol. An in vivo eye irritation test in rabbits was conducted with the undiluted test substance VBE (EC 444-010-7). The test material did cause significant eye irritation. However, the degree of eye irritation observed would result in a GHS classification of EDI Cat. 2/2A (based on Table 3.3.2, page 140 of GHS v.7). These results support the position that Ethyl Vanillyl Alcohol is likely to be a GHS Cat. 2 eye irritant.

o Vanillyl Alcohol (CAS 498-00-0) is only 1-carbon different in structure from Ethyl Vanillyl Alcohol. In addition, Vanillin (CAS 121-33-5) is structurally similar to Ethyl Vanillyl Alcohol, with the key difference that Vanillin (CAS 121-33-5) is an aldehyde while Ethyl Vanillyl Alcohol is an alcohol.

Since aldehydes are typically more reactive than alcohols, it would be expected that Vanillin (CAS 121-33-5) would be more irritating than Ethyl Vanillyl Alcohol. According to the ECHA website, both Vanillyl Alcohol (CAS 121-33-5) and Vanillin (CAS 121-33-5) are classified as a Cat. 2 eye irritant under the EU CLP. Again, this supports the conclusion to classify Ethyl Vanillyl Alcohol as EDI Cat. 2.

Due to the results of the existing in vitro assays on the substance Ethyl Vanillyl Alcohol (EC 674-192-5 / CAS 4912-58-7) and the justification above based on the weight of evidence approach, conducting an in vivo assay is not justified. The substance Ethyl Vanillyl Alcohol (EC 674-192-5 / CAS 4912-58-7) can be classified as EDI Category 2, according to the EU CLP and GHS classification.