Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Endpoint summary

Description of key information

Skin Irritation / Corrosion

Skin corrosion in vitro. Key CRL 2020

OECD 431

The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Skin irritation in vitro. Key CRL 2020

OECD 439

The test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Skin irritation / corrosion in vivo. Key CRL 2020

OECD 404

Based on the results of the test item does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Eye Irritation

Eye irritation in vitro. Key CRL 2020

OECD 437

In conclusion, since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.  

Eye irritation in vivo. Key CRL 2020

OECD 405

Based on these results, the test item does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

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:

12 November 2019 - 28 January 2020

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)

Version / remarks:

adopted 18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

31 May 2008

Deviations:

no

Principles of method if other than guideline:

DEviation:
Cell Viability Measurement
• The start time for the MTT incubation was not noted correctly. The correct time cannot be
traced.
Evaluation: The responses for the negative and positive control indicated that an
appropriate incubation time was used. Therefore this deviation has no impact on the study
result..

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
EC Number: 951-458-1
Substance name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Test item handling: Use amber glassware or wrap container in aluminum foil

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Reconstructed human epidermis

Source strain:

not specified

Details on animal used as source of test system:

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Model used: EpiDerm Reconstucted Human Epidermmis
Tissue batch numbers: 30928 and 32126
Date of initiation of testing: 25 November 2019
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM.

TEST ITEM PREPARATION
No correction was made for the purity/composition of the test item. The liquid test item was applied undiluted (50 µL) directly on top of the tissue. To protect the test item from light glassware was wrapped in tin-foil.

TEMPERATURE USED FOR TEST SYSTEM
Temperature used during treatment / exposure: 36.5 - 37.2°C

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data, these deviations are considered not to affect the study integrity.

APPLICATION/TREATMENT OF THE TEST ITEM
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 µL of the undiluted test item was added into the 6-well plates on top of the skin tissues. In addition, since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT.For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data.

REMOVAL OF TEST MATERIAL AND CONTROLS
Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm
- Filter/Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

The amount of extracted formazan was determined spectrophotometrically in triplicate.

Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item:
The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the
exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

Test for Reduction of MTT by the Test Item:
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline.


CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Fresh tissues / killed tissues: Killed tissues
N. of replicates : since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT.

Procedure used to prepare the killed tissues
Freeze-killed. Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to ≤-15°C. The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 mL DMEM. Further use of killed tissues was similar to living tissues. DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM, serum-free.

Cell Viability Measurement
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature.


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.


ACCEPTABILITY CRITERIA
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8).

b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.

c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD. All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.


INTERPRETATION
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Table 1 presents the data interpretation and optional sub-categorisation in case a test item will be corrosive
See Table 1: Data interpretation and sub-categorisation of test items in 'Any other information on materials and methods' incl. tables

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL liquid test item (undiluted)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 normal solution

Duration of treatment / exposure:

3-minute and 1-hour treatment/exposures

Number of replicates:

Two (two tissues per exposure period and two tissues each for the positive and negative controls)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute treatment with the test item

Value:

80

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour treatment with the test item

Value:

84

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:

Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.  

In addition to the normal 3-minute and 1-hour procedure, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with DMEM. The non-specific reduction of MTT by the test item was 0.63% and 1.62% of the negative control tissues after 3 minutes and 1 hour respectively.  

The mean absorption at 570 nm measured after treatment with the test item and controls are presented in Appendix 1, Table 1. The individual OD570 measurements are presented in Appendix 2.  

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative Control	1.787	2.106	1.946	±	0.226	1.837	1.986	1.946	±	0.226
Test Item^(1)	1.565	1.559	1.562	±	0.004	1.741	1.480	1.562	±	0.004
Positive Control	0.187	0.178	0.183	±	0.007	0.129	0.187	0.183	±	0.007
SD = Standard deviation
Duplicate exposures are indicated by A and B.
^(1)The test item values are corrected for the non-specific MTT reaction (0.63 and 1.62 at the 3 minute and 1 hour treatment, respectively).

The individual OD570 measurements are presented in the following table (attached in Appendix 2):

	3-minute application (OD570)		1-hour application (OD570)
	A	B	A	B
Negative control
OD570measurement 1	1.8243	2.1438	1.9075	2.0896
OD570measurement 2	1.8348	2.1432	1.8636	1.9975
OD570measurement 3	1.8304	2.1618	1.8709	2.0008
Test item
OD570measurement 1	1.6544	1.6057	1.8401	1.5761
OD570measurement 2	1.6024	1.6116	1.7981	1.5495
OD570measurement 3	1.6047	1.6261	1.8085	1.5357
Test item on freeze killed tissue
OD570measurement 1	0.1645	0.1743	0.1560	0.1557
OD570measurement 2	0.1623	0.1689	0.1554	0.1519
OD570measurement 3	0.1642	0.1859	0.1552	0.1544
Non - treated freeze killed tissue
OD570measurement 1	0.1214	0.2338	0.1231	0.1284
OD570measurement 2	0.1207	0.2157	0.1203	0.1251
OD570measurement 3	0.1228	0.1324	0.1212	0.1249
Positive control
OD570measurement 1	0.2307	0.2255	0.1762	0.2416
OD570measurement 2	0.2301	0.2177	0.1702	0.2284
OD570measurement 3	0.2312	0.2204	0.1701	0.2209
OD = Optical density
Duplicate exposures are indicated by A and B.

The following table (Table 2, Appendix 1) shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues:

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	80	84
Positive control	9.4	8.3

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 80% and 84% respectively.  Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit≤2.8) and the laboratory historical control data range (See Appendix 3). The mean relative tissue viability following the 1-hour exposure to the positive control was 8.3%.  

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was≤15%, indicating that the test system functioned properly (Appendix 1, Table 3).

	3 minute	1 hour
Negative control	15	7.5
Test item	0.4	15
Positive control	5.1	31
CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item was a light orange liquid. The test item was applied undiluted (50 µL) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 8.3% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤15%, indicating that the test system functioned properly.

Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.  

In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test item was 0.63% and 1.62% of the negative control tissues after 3 minutes and 1 hour respectively.  

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 80% and 84%, respectively.  Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February 2020 - 10 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, OPPTS 870.2500 Acute Dermal Irritation

Version / remarks:

part B

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines including the most recent revisions

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum foil
Substance Name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Species:

rabbit

Strain:

New Zealand White

Remarks:

SPF-Quality

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River France, L’Arbresle, Franc
Age at study initiation: approximately 11-22 weeks old
Weight at study initiation: 2359 to 3924 g

Justification for Test Animal Species
The New Zealand White rabbit was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW).

Housing:
On arrival and following assignment to the study, animals were housed individually in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. The room in which the animals were kept was documented in the study records. Each cage was clearly labeled.

Diet:
Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Granovit AG, Kaiseraugst, Swizerland) was provided once daily throughout the study. In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was available during the study period. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Acclimation period:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Animal Identification
At study assignment, each animal was identified using an ear mark with indelible ink.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. The disposition of all animals was documented in the study records.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) except when interrupted by study
procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 19°C
- Humidity: 54 to 55%
- Air changes: Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES:
Study Initiation Date: 14 Feb 2020
Initiation of Dosing: 19 Feb 2020
Completion of In-life: 01 Mar 2020
Experimental Start Date: 19 Feb 2020
Experimental Completion Date: 01 Mar 2020

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Each animal was treated by dermal application of 0.5 mL of the test item.

Duration of treatment / exposure:

4 hours

Observation period:

7 days

Number of animals:

3 animals

Details on study design:

TEST SITE
Area of exposure: approximately 150 square centimeters (10x15 cm)
Type of wrap if used: The test item was applied to the skin of one flank, using a metalline patch of 2x3 cm. The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage.

REMOVAL OF TEST SUBSTANCE
Four hours after the application, the dressing was removed and the skin cleaned of residual test item using tap water.


OBSERVATION TIME POINTS
The skin reactions were assessed at approximately 1, 24, 48 and 72 hours and 7 days after the removal of the dressings and test item. The irritation scores and a description of all other (local) effects were recorded.

SCORING SYSTEM:
The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:
To facilitate scoring, the treated skin area of one animal was re-clipped at least 3 hours before the observations.

Erythema and eschar formation:
0 No erythema
1 Very slight erythema (barely perceptible)
2 Well-defined erythema
3 Moderate to severe erythema
4 Severe erythema (beef redness) *
*. Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema (= 4) is given.

Oedema formation:
0 No oedema
1 Very slight oedema (barely perceptible)
2 Slight oedema (edges of area well-defined by definite raising)
3 Moderate oedema (raised approximately 1 millimeter)
4 Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure)



Histopathology
No histopathology was performed since sufficient information was obtained within this study.

Terminal Procedures
After the final observation and weighing, the animals were euthanized according to laboratories Standard Operating Procedures.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Irritation
Four hours exposure to C894 resulted in very slight erythema in the treated skin areas of the rabbits, which had resolved within 72 hours for two animals and 7 days for one animal. Very slight oedema was only seen in one animal which had resolved within 24 hours.

Coloration / Remnants
Remnants of the test item were present on the skin of all animals on Days 1 and/or 2.

Toxicity / Mortality
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Full data tables are in Appendix 1, appended to 'Attached Background Material'

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results the test item does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this primary skin irritation study was to assess the possible irritation or corrosion potential of a single dose of the test item when administered to the intact skin of rabbits.  

The study was carried out in compliance with the guidelines described in:  

• OECD No. 404 (2015) "Acute Dermal Irritation / Corrosion".

• EC No 440/2008, part B: "Acute Toxicity: Dermal Irritation/Corrosion".

• EPA, OPPTS 870.2500 (1998), "Acute Dermal Irritation".

• JMAFF Guidelines (2000), including the most recent revisions.

Three rabbits were exposed to 0.5 mL of the test item by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours and 7 days after exposure.

Exposure to the test item resulted in very slight erythema in the treated skin areas of the rabbits, which had resolved within 72 hours for two animals and 7 days for one animal. Very slight oedema was only seen in one animal which had resolved within 24 hours. Remnants of the test item were present on the skin of all animals on Days 1 and/or 2.

Based on these results the test item does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 December 2019 - 07 April 2020

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)

Version / remarks:

adopted 18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test "

Version / remarks:

Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Deviations:

no

Principles of method if other than guideline:

Deviations
Cell Viability Measurement
- The study plan states that extraction with isopropanol should be for 70 +/- 3 hours. However, the extraction was performed for 66 hours, which is not according to the study plan.
- Evaluation: According to Episkin validation model, formazan extraction should be performed for 18 to 72 hours. In addition, all criteria are met. Therefore, the assay was still valid and the 66 hours extraction did not influence the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Test item handling: Use amber glassware or wrap container in aluminum foil
EC number: 951-458-1
Substance name Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Reconstructed human epidermis

Source strain:

not specified

Details on animal used as source of test system:

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.SOURCE ANIMAL

Justification for test system used:

Recommended test system in international guidelines (OECD and EC)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 20 EKIN 002)
- Tissue batch number(s): 19 EKIN 043, 19 EKIN 045 and 19 EKIN 047
- Date of initiation of testing: 07 Jan 2020
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with 2 mL pre-warmed Maintenance Medium for 18 - 24 hours at 37°C. Maintenance medium and Assay medium are supplied by Skinethic Laboratories, Lyon France.

TEMPERATURE USED FOR TEST SYSTEM
- All incubations were carried out in a humid atmosphere (80 - 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. Temperature and humidity was continuously monitored throughout the experiment.
- Subsequently the skin tissues were incubated for 42 ± 1 hours at 37°C.

APPLICATION/TREATMENT OF THE TEST ITEM
The test was performed on a total of 3 tissues per test item together with a negative control and positive control.
At least 25 µL of the undiluted test item was added into the 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. Negative and positive controls can be shared with parallel studies.
Test items that reduce MTT non-specific require in addition to the normal procedure, 3 killed test item treated tissues and 3 killed untreated tissues were used for the cytotoxicity evaluation with MTT.

REMOVAL OF TEST MATERIAL AND CONTROLS
All incubations were carried out in a humid atmosphere (80 - 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage could have occurred due to opening and closing of the incubator door. Any variation to these conditions was evaluated and maintained in the raw data.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).
- Incubation time: Living epidermis transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ± 1 hours.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter/ Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

Test for the Interference of the Test Item with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
Test for possible direct MTT reduction and colour interference by the Test Item:
The test item was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20221636).
Because solutions did turn blue / purple and/or a blue / purple precipitate was observed it was concluded that the test item did interfere with the MTT endpoint.


CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: killed tissues
- N. of replicates : three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT.
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
Three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT with an exposure period of 15 ± 0.5 minutes at room temperature.
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered irritant in the skin irritation test if:
a) The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
a) The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Table 1 presents the data interpretation of test items.

See Table 1: Data interpretation of test items in 'Any other information on materials and methods' incl. tables



PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 µL liquid test item (undiluted)
To protect the test item from light glassware was wrapped in tin-foil.

NEGATIVE CONTROL
- Amount applied: 25 µL Phosphate buffered saline (PBS).

POSITIVE CONTROL
- Amount applied: 25 µL
- Concentration: 5% (aq) Sodium dodecyl sulfate (SDS) (re-spread after 7 minutes contact time)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

The test is performed on a total of 3 tissues per test item together with a negative control and positive control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 ± 0.5 minutes treatment with the test item

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The test item was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20221636).

Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.  

In addition to the normal procedure, three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was -1.5% of the negative control tissues. The net OD of the treated killed tissues was not subtracted from the ODs of the test item treated viable tissues.

The mean absorption at 570 nm measured after treatment with the test item and controls are presented in the below table:

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	3
Test item	98	13
Positive control	7.8	2.9

The mean absorption at 570 nm measured after treatment with the test item and controls are presented below:

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.109	1.06	1.121	1.096	±	0.032
Test item	0.972	1.021	1.241	1.078	±	0.143
Positive control	0.054	0.115	0.09	0.086	±	0.031

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.046). Isopropanol was used

to measure the background absorption.

The individual OD570 measurements are presented below:

	A (OD570)	B (OD570)	C (OD570)
Negative control
OD570 measurement 1	1.1479	1.1062	1.1677
OD570 measurement 2	1.1623	1.1058	1.1671
Test item on viable tissue
OD570 measurement 1	1.0429	1.0633	1.2939
OD570 measurement 2	0.9945	1.0706	1.2812
Test item on killed tissue
OD570 measurement 1	0.1189	0.0782	0.0847
OD570 measurement 2	0.1128	0.1291	0.0918
Non treated killed tissue
OD570 measurement 1	0.1819	0.1067	0.0815
OD570 measurement 2	0.1608	0.1029	0.0819
Positive control
OD570 measurement 1	0.1027	0.1628	0.1292
OD570 measurement 2	0.0954	0.1598	0.1441

The table below shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 98%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.8%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Appendix 3). The standard deviation value of the percentage viability of three tissues treated identically was ≤ 13%, indicating that the test system functioned properly.

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	3
Test item	98	13
Positive control	7.8	2.9

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the test item for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item was a light orange liquid. The test item was applied undiluted (25 µL), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was -1.5% of the negative control tissues. The net OD of the treated killed tissues was not subtracted from the ODs of the test item treated viable tissues.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 98%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 7.8% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 13%, indicating that the test system functioned properly.

In conclusion, the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

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:

06 November 2019 - 13 January 2020

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)

Version / remarks:

adopted October 09, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
EC Number: 951-458-1
Substance name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Test item handling: Use amber glassware or wrap container in aluminum foil

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

No correction was made for the purity/composition of the test item. To protect the test item from light, tubes wrapped in tin-foil were used. The test item was tested neat.

Amount
750 µL of the negative control, positive control (Ethanol)
Test item (excessive amount)

Duration of treatment / exposure:

10 ± 1 minutes at 32 ± 1°C.

Experimental dates:
Start: 26 November 2019
End: 26 November 2019

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes at 32 ± 1°C

Number of animals or in vitro replicates:

3 per treatment group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
3 per treatment group

NEGATIVE CONTROL USED
A negative control, physiological saline (Merck KGaA, Darmstadt, Germany) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED
The positive control was Ethanol (Merck KGaA, Darmstadt, Germany).

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item (excessive amount) was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.

Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

METHODS FOR MEASURED ENDPOINTS:

- Corneal opacity:
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:

Opacity = ((I0/I) - 0.9894) / 0.0251

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was
calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader).
Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:

In vitro score range UN GHS
≤ 3 No Category

> 3; ≤ 55 No prediction can be made

>55 Category 1

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

3

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: permeablity score

Run / experiment:

1

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: permeablity score

Run / experiment:

2

Value:

-0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: permeablity score

Run / experiment:

3

Value:

-0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item was tested neat.

Table 1 of Appendix 1 summarizes the opacity, permeability and in vitro irritancy scores of C894 and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2 - 5.

The individual in vitro irritancy scores for the negative controls ranged from 2.4 to 2.9. The corneas treated with the negative control item were clear after the 10 minutes of treatment.
The individual positive control in vitro irritancy scores ranged from 40 to 54 (Appendix 2, Table 5). The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from 1.1 to 2.4 and permeability values ranging from -0.001 to 0.003. The corneas were translucent after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 1.1 to 2.4 after 10 minutes of treatment with the test item.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 48 and within two standard deviations of the current historical positive control mean (Appendix 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.6 after 10 minutes of treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since C894 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of C894 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for 10 minutes.  

The study procedures described in this report were based on the most recent OECD guideline.

Batch 11187 of the test item was a light orange liquid. The test item was applied as it is (excessive amount) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 48 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.6 after 10 minutes of treatment.  

In conclusion, since C894 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.  

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 March 2020 - 04 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, part B: "Acute Toxicity: Eye Irritation/Corrosion"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines including the most recent revisions

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum foil
Substance Name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Chemical name (IUPAC): 2,2-bis[({3-[methyl(phenyl)amino]propanoyl}oxy)methyl]butyl 3-[methyl(phenyl)amino]propanoate
EC number: 951-458-1
Molecular formula: EtC(CH2OCOCH2CH2NMePh)3
Molecular weight: 617.79 g/mol

Species:

rabbit

Strain:

New Zealand White

Remarks:

SPF-Quality

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
Source: Charles River France, L’Arbresle, France
Age at study initiation: approximately 12-14 weeks old
Weight at study initiation: 2350 to 2987 g

Housing
On arrival and following assignment to the study, animals were housed individually in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. The room in which the animals were kept was documented in the study records. Each cage was clearly labeled.

Diet
Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Granovit AG, Kaiseraugst, Swizerland) was provided once daily throughout the study. In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was available during the study period.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Acclimation period:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing

Animal Enrichment
For psychological/environmental enrichment, animals were provided with shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Pre-emptive Pain Management
One hour prior to instillation of the test item, buprenorphine (Buprenodale®) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test item, two drops of the topical anaesthetic 0.5% proparacaine hydrochloric ophthalmic solution (Tetracaine eye drops®) were applied to both eyes.


ENVIRONMENTAL CONDITIONS
- Temperature: The actual daily mean temperature during the study period was 19°C (target temperatures of 18 to 24°C)
- Humidity: The actual daily mean relative humidity was 51 to 54% (relative target humidity of 40 to 70%)
- Air changes: Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod: 12-hour light/12-hour dark cycle


IN-LIFE DATES:
Study Initiation Date: 25 Mar 2020
Initiation of Dosing: 30 Mar 2020
Completion of In-life: 10 Apr 2020
Experimental Start Date: 30 Mar 2020
Experimental Completion Date: 10 Apr 2020


Justification for Test System and Number of Animals
The New Zealand White rabbit was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals were based on the test guidelines.
The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified using an ear mark with indelible ink.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. The disposition of all animals was documented in the study records.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
Amount applied: 0.1 mL of test item

ADMINISTRATION OF TEST ITEM
Each animal was treated by instillation of 0.1 mL of the test item, in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test item. The other eye remained untreated and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage.
In order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection.

Justification of Route and Dose Level
The ocular route was selected because the test item may accidentally come in contact with the eyes during manufacture, handling and/or use. The dose level was based on the test guidelines.

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 animals

Details on study design:

REMOVAL OF TEST SUBSTANCE
No washing reported.

SCORING SYSTEM:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded.
The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

Corneal Irritation
Opacity: degree of density (area most dense taken for reading)
0 No ulceration or opacity (may include slight dulling of normal luster)
1 Scattered or diffuse areas of opacity, details of iris clearly visible
2 Easily discernible translucent area, details of iris slightly obscured
3 Nacreous area, no details of iris visible, size of pupil barely discernible
4 Opaque cornea, iris not discernible through the opacity

Area of cornea involved:
0 No ulceration or opacity
1 One quarter or less but not zero
2 Greater than one quarter, but less than half
3 Greater than half, but less than three quarters
4 Greater than three quarters, up to whole area

Iris
0 Normal
1 Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or combination thereof, iris still reacting to light (sluggish reaction is positive)
2 No reaction to light, hemorrhage, gross destruction (any or all of these)

Conjunctival irritation
Redness (refers to palpebrae and sclera, excluding cornea and iris):
0 Blood vessels normal
1 Some blood vessels definitely hyperaemic (injected)
2 Diffuse, crimson color, individual vessels not easily discernible
3 Diffuse beefy red

Chemosis (refers to lids and/or nictitating membranes):
0 No swelling
1 Any swelling above normal (includes nictitating membranes)
2 Obvious swelling with partial eversion of lids
3 Swelling with lids about half closed
4 Swelling with lids more than half closed

Discharge:
0 No discharge (may include small amounts observed in inner canthus of normal animals)
1 Any amount different from normal and/or lacrimation
2 Discharge with moistening of the lids and hairs just adjacent to lids
3 Discharge with moistening of the lids and hairs (considerable area around the eye)

Where standard lighting was considered inadequate for observing minor effects, eye examinations were performed using an ophthalmic examination lamp.


TOOL USED TO ASSESS SCORE: fluorescein


IN-LIFE PROCEDURES, OBSERVATIONS AND MEASUREMENTS
Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Toxicity
Observations for toxicity were performed once daily throughout the study.

Body Weights
Animals were weighed individually on Day 1 (pre-dose) and on the day of the final observation.

Terminal Procedures
After the final observation, the animals were euthanized according to laboratories Standard Operating Procedures.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Discharge

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

conjunctivae score

Remarks:

Discharge

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

conjunctivae score

Remarks:

Discharge

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 hours

Irritant / corrosive response data:

Irritation
Instillation of approximately 0.1 mL of C894 into one eye of each of three rabbits resulted in
irritation of the conjunctivae, which consisted of redness and discharge. The irritation had
completely resolved within 48 hours in two animals and within 72 hours in one animal.
No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2%
fluorescein 24 hours after test item instillation revealed no corneal epithelial damage.
8.2. Coloration / Remnants
Remnants of the test item were present on the outside of the eyelids in one animal on Day 1.
This is considered to be due to innate protective physiological mechanisms of the eye against
entry of foreign compounds.
8.3. Toxicity / Mortality
No signs of systemic toxicity were observed in the animals during the test period and no
mortality occurred.

For detailed results see Appendix 1 (appended to attached background material)

Irritation

Instillation of approximately 0.1 mL of C894 into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness and discharge. The irritation had completely resolved within 48 hours in two animals and within 72 hours in one animal.

No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test item instillation revealed no corneal epithelial damage.

Coloration / Remnants

Remnants of the test item were present on the outside of the eyelids in one animal on Day 1. This is considered to be due to innate protective physiological mechanisms of the eye against entry of foreign compounds.

Toxicity / Mortality

No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results, C894 does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this acute eye irritation study was to assess the possible irritation or corrosion potential when a single dose of C894 was placed in the conjunctival sac of the rabbit eye.  

The study was carried out in compliance with the guidelines described in:  

• OECD No.405 (2017) "Acute Eye Irritation / Corrosion".

• EC No 440/2008, part B: "Acute Toxicity: Eye Irritation/Corrosion".

• EPA, OPPTS 870.2400 (1998), "Acute Eye Irritation".

• JMAFF Guidelines (2000), including the most recent revisions.

Single samples of 0.1 mL of C894 were instilled into one eye of each of three rabbits.

Observations were made 1, 24, 48 and 72 hours after instillation.  

Instillation of the test item resulted in irritation of the conjunctivae, which consisted of redness and discharge. The irritation had completely resolved within 48 hours in two animals

and within 72 hours in one animal.

Based on these results, C894 does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Studies conducted to recognised testing guidelines with GLP certification.

Justification for classification or non-classification

The test item does not have to be classified and has no obligatory labelling requirement for skin irritation or eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).