Registration Dossier

Administrative data

Description of key information

In vitro skin corrosion, Reconstructed human epidermis test method (OECD 431): not corrosive

In vitro skin irriation, Reconstructed human epidermis test method (OECD 439): not irritating

In vitro eye irritation, Reconstructed human cornea-line epithelium model (OECD 492): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 - 22 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU-Method B.40 BIS (in vitro skin corrosion: human skin model test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Source strain:
other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200-SCT)
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST METHOD:
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS:
Upon receipt, tissues were transferred into 6-well plates containing 900 µL pre-warmed assay medium per well and preincubated in a humidified incubator for at least 1 h (37 ± 1 °C, 5% CO2) before use.

INCUBATION CONDITIONS (INCUBATOR):
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: maximum

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE :
- Model used: EpiDermTM EPI-200-SCT
- Tissue batch number(s): 25822
- Delivery date: 20 June 2017

TEMPERATURE USED FOR TEST SYSTEM :
- Temperature used during treatment / exposure: room temperature (3 min exposure) and 37 +/- 1 °C, 5.0 +/- 0.5% CO2 (1 h exposure)
- Temperature of post-treatment incubation (if applicable): 37 +/- 1 °C, 5.0 +/- 0.5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS :
-Volume and number of washing steps: thoroughly rinsed with Dulbecco's phosphate-buffered saline and blotted with sterile cellulose tissue
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

CELL VIABILITY MEASUREMENTS:
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, tissues were incubated in 300 µL pre-warmed MTT solution for 3 h at 37 ± 1 °C and 5% CO2. After aspiration of the MTT solution, tissues were washed 3 times in phosphate buffered saline followed by tissue drying. Extraction of the formazan product was carried out in 2 mL isopropanol. The optical density was measured at 570 nm wave length in a plate spectrophotometer.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE :
- MTT concentration: 5 mg/mL
- Incubation time: 3 h, 37 +/- 1 °C, 5 +/- 0.5 % CO2
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive to skin if 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 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%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8N
Duration of treatment / exposure:
3 and 60 min
Duration of post-treatment incubation (if applicable):
1 h
Number of replicates:
The test was performed in duplicates for each test or control group and treatment period (3 and 60 min).
Species:
other: in vitro system
Type of coverage:
other: in vitro system
Irritation / corrosion parameter:
% tissue viability
Remarks:
test item
Run / experiment:
3 min
Value:
99.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
test item
Run / experiment:
60 min
Value:
107.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
positive control
Run / experiment:
3 min
Value:
19.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
positive control
Run / experiment:
60 min
Value:
7.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no, as assessed in a suitable pre-experiment
- Colour interference with MTT: no, as assessed in a suitable pre-experiment

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD 1.8 (3 min) and 1.6 (1 h) (criterion: OD ≥ 0.8 and ≤ 2.8)
- Acceptance criteria met for positive control: yes, % tissue viability after 1 h: 7.8 (criterion: < 15%)
- Range of historical values if different from the ones specified in the test guideline: values for negative control and for positive control were within the range of historical data of the test facility

The values of the negative control met the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals, thus showing the quality of the tissues. The positive control has met the validity criterion too, thus ensuring the validity of the test system. For these reasons, the result of the test is considered valid.

Table 1: Absorbance values blank isopropanol (OD 570nm)

 

Replicate

1

2

3

4

5

6

 

Mean 0.038

Absorbance

0.038

0.037

0.039

0.039

0.038

0.038

Replicate

7

8

9

10

11

12

Absorbance

0.039

0.038

0.038

0.039

0.038

0.037

 

Table 2: Absorbance Values (OD 570nm) for negative control, test item and positive control

 

Incubation

Negative Control

Test Item

Positive Control

 

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Tissue 1

Tissue 2

 

3 min

1.797

1.826

1.830

1.822

0.380

0.385

1.853

1.786

1.761

1.813

0.381

0.384

1.791

1.776

1.771

1.818

0.378

0.385

 

1 h

1.602

1.600

1.741

1.720

0.167

0.157

1.596

1.607

1.767

1.679

0.165

0.156

1.597

1.618

1.722

1.686

0.166

0.154

 

Table 3: Mean Absorbance Values of the 3 min Experiment

 

Designation

Negative Control

Test Item

Positive Control

Mean – blank (tissue 1)

1.776

1.749

0.342

Mean – blank (tissue 2)

1.758

1.780

0.347

Mean of the two tissues

1.767

1.764

0.344

RSD

0.7%

1.2%

1.0%

 

Table: 4: Mean Absorbance Values of the 1 h Experiment

 

Designation

Negative Control

Test Item

Positive Control

Mean – blank (tissue 1)

1.560

1.705

0.128

Mean – blank (tissue 2)

1.570

1.657

0.118

Mean of the two tissues

1.565

1.681

0.123

RSD

0.5%

2.0%

6.0%

 

  Table 5: % Tissue Viability

 

Test Item

Positive Control

Incubation

99.9 %

19.5 %

3 min

107.4 %

7.8 %

1 h

 

Table 6: Historical Data

 

Parameter

Optical Density Negative Control

Optical Density Negative Con- trol

% Tissue viability Positive Control

% Tissue viability Positive Control

Incubation Time

3 min.

1 h

3 min.

1 h

Mean

1.956

1.905

25.1 %

12 %

Standard Deviation

0.275

0.220

6.8 %

3.8 %

Range

1.197 - 3.077

1.377 - 2.571

9.6 - 57.3 %

4.1 - 24.2 %

Study17051601G820

 

1.767

 

1.565

 

19.5

 

7.8

Interpretation of results:
other: not corrosive
Conclusions:
There is regulatory acceptance in the EU that a substance can be considered corrosive based on a positive result in the human epidermis model test (OECD 431). Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant. Therefore, the result obtained needs to be supported by additional data in order to conclude the hazard assessment and determination of the classification and labelling.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 - 11 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Source strain:
other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200-SIT)
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST METHOD:
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE:
- Model used: EpiDermTM EPI-200-SIT
- Tissue batch number(s): 25835
- Delivery date: 08 Aug 2017

ADAPTATION TO CELL CULTURE CONDITIONS (PRE-INCUBATION):
For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells. The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1 °C and 5 ± 0.5% CO2 for 1 h. After 1 h pre-incubation, the other 3 wells of each plate were filled with fresh assay medium (0.9 mL). All 6-well-plates were set into the incubator at 37 ± 1 °C and 5.0 ± 0.5% CO2 for 19 h.

TREATMENT AND MEDIUM RENEWAL:
Tissues were dosed in 1-minute intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 min at 37 ± 1 °C and 5.0 ± 0.5% CO2. 1 h after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute intervals. After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). Then, the tissues were set in the incubator for 23.5 h at 37 ± 1 °C and 5.0 ± 0.5% CO2.

After post-incubation, the tissues were removed from the incubator and shaken for 5 min (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-wellplate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 h 5 min for post-incubation at 37 ± 1 °C and 5.0 ± 0.5% CO2.

CELL VIABILITY MEASUREMENTS:
After a total incubation time of 42 h 35 min, a 24-well-plate was prepared with 300 μL freshly prepared MTT-solution in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 h at 37 ± 1 °C and 5.0 ± 0.5% CO2. After this time, the MTT-solution was aspirated and replaced by Dulbecco's phosphate-buffered saline buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken (120 rpm) for 2 h at room temperature before they were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly
mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well plate
which was read in a plate spectrophotometer at 570 nm.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL
- The test substance is considered to be corrosive or irritating to skin if the viability is less than or equal to 50% after 1 h exposure
- The test substance is considered to be not irritating to skin if the viability is greater than 50% after 1 h exposure
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 26 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 h 35 min
Number of replicates:
The test was performed in triplicates for each test or control group.
Species:
other: in vitro system
Type of coverage:
other: in vitro system
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of all 3 test item tissues
Value:
100.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of all 3 positive control tissues
Value:
2.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no, as assessed in a suitable pre-experiment
- Colour interference with MTT: no, as assessed in a suitable pre-experiment

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD of negative control 1.6 (criterion: ≥ 0.8 and ≤ 2.8)
- Acceptance criteria met for positive control: yes, % tissue viability 2.2 (criterion: ≤ 20%)
- Acceptance criteria met for variability between replicate measurements: yes, SD 9.7% (negative control), 0.2% (positive control), 5.4% (test item) (criterion: ≤ 18%)
- Range of historical values if different from the ones specified in the test guideline: yes, all values for negative control and for positive control were within the range of historical data of the test facility

Table 1: Absorbance Values negative control, test item and positive control (OD 570 nm)

 

Designa- tion

Measure- ment

 

Negative Control

(2Z)-4-

(octadecylamino)-4- oxo-2-butenoic acid

 

Positive Control

Tissue 1

1

1.829

1.538

0.072

2

1.879

1.654

0.072

Tissue 2

1

1.621

1.635

0.074

2

1.580

1.755

0.071

Tissue 3

1

1.559

1.745

0.078

2

1.562

1.797

0.077

 

Table 2: Mean Absorbance Values

 

 

Designation

 

Negative Control

(2Z)-4-

(octadecylamino)-4- oxo-2-butenoic acid

 

Positive Control

Mean – blank (tissue 1)

1.815

1.557

0.033

Mean – blank (tissue 2)

1.562

1.656

0.034

Mean – blank (tissue 3)

1.522

1.732

0.039

Mean of the three tissues

 

1.633

 

1.648

 

0.035

Table 3: % Tissue Viability

 

Designation

(2Z)-4-(octadecylamino)- 4-oxo-2-butenoic acid

Positive Control

% Tissue viability (tissue 1)

95.3%

2.0%

% Tissue viability (tissue 2)

101.4%

2.1%

% Tissue viability (tissue 3)

106.1%

2.4%

% Tissue viability (mean)

100.9%

2.2%

± SD of mean tissue viability (%)

5.4%

0.2%

 

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
There is regulatory acceptance in the EU that a substance can be considered not irritating to skin based on an appropriate result in the human epidermis model test (OECD 439).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June - 26 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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 09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Species:
human
Strain:
other: EpiOcular™
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. The cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular™ tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years and for a great variety of chemicals.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

The test was performed on a total of 2 tissues per dose group.
Duration of treatment / exposure:
6 h (37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity)
Duration of post- treatment incubation (in vitro):
post-exposure immersion: 25 min (room temperature)
post-treatment incubation: 18 h (37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity)
Number of animals or in vitro replicates:
duplicate tissues were investigated
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ tissues (OCL-200-EIT; MatTek, Lot No.: 27046)

MTT-REDUCING PRE-EXPERIMENT
To test for direct MTT reduction of the test item, 50.9 mg of the test item were added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2, 80 – 100 % relative humidity for 3 h. 1 mL of MTT solution plus 50 µL of demineralised water was used as negative control. The MTT solution did not change its colour. Therefore, direct MTT reduction had not taken place and no data correction was necessary.

COLOURING POTENTIAL PRE-EXPERIMENT
49.6 mg of the test item were added to 2 mL isopropanol and incubated in 6-well plates on an orbital shaker for 2 h at room temperature. The test item was not completely soluble in isopropanol. Therefore, the test item solution was centrifuged for 30 s at 16,000g. Then, two 200 µL aliquots of the supernatant of the resulting solution and two 200 µL aliquots of neat isopropanol were transferred into a 96-well plate and the optical density (OD) was measured with a plate reader at 570 nm. After subtraction of the mean OD for isopropanol, the mean OD of the test item solution was 0.16 (>0.08). The test item was possibly interacting with the photometrical measurement and an additional test on colourant controls was performed. The additional test was performed in order to evaluate the amount of colour bound to the tissues. The test item was applied to two additional tissues (= colourant controls) and the test was performed in the same way as described for the main test, but no MTT assay was performed. Instead of 300 µL MTT solution, 300 µL assay medium were used. The bound colour was extracted and the absorbance of the isopropanol extract was measured in the same way as in the MTT assay for coloured test items. As the colourant control result was ≤ 50% compared to the negative control, a data correction procedure could be performed.

EXPERIMENTAL PROCEDURE
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1% CO2, 80 – 100% relative humidity for 1 h. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity for 16 h.
After overnight incubation, the tissues were pre-wetted with 20 µL Dulbecco`s Phosphate Buffered Saline (DPBS) buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity for 29 min. After that, 50 µL of the controls and 50 mg of the test item were applied in duplicate in one minute intervals. After dosing the last tissue, all plates were transferred into the incubator for 6 h at 37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one minute intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plates for 25 min post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 h at 37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity.
A 24-well-plate was prepared with 300 µL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 min at 37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 h at room temperature, protected from light. The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well- plate. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

PREDICTION MODEL
The ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with demineralised water. The test item is considered to be irritant to the eye if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant if relative tissue viability is higher than 60%.

ACCEPTANCE CRITERIA
The test meets acceptance criteria if:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%.

CALCULATIONS
Note: The corrected optical density (OD) value of the negative control corresponds to 100% viability.

% viability = [ ODcorrected test item/positive control / ODcorrected negative control ] x 100

% viability (colourant control) = [ ODcorrected test item at colourant control / ODcorrected negative control ] x 100
NB: "% viability (colourant control)" refers to the negative control in the same experiment. THe value for "% viability (colourant control)" was subtracted from "% viability" of the main test.
Irritation parameter:
other: mean relative viability (%)
Run / experiment:
6 h
Value:
103.7
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: The negative control OD (main test: 2.0; additional test: 1.8) was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (main test: 38.2%).
- The difference of rel. absorbance between the two relating tissues of a single item is < 20% (main test: 1.6% for negative control, 5.0% for positive control, 9.7% for test item; additional test: 11.5% for negative control, 0% for test item).

Measured optical densities and calculated values are summarised in the a separate pdf document (OECD 492 results.pdf)

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
After treatment with the test item, the mean value of relative tissue viability was 103.7 %. This value is well above the threshold for eye irritation potential (≤ 60%). The test item is, therefore, considered not to be irritating to eyes in the in vitro EpiOcular Eye Irritation Test (EIT).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro skin corrosion:

The skin corrosion potential of the test substance was determined by an in vitro skin corrosion test using a reconstructed human skin model according to OECD guideline 431 and in compliance with GLP (LAUS, 2017a). Skin corrosion potential was identified by reduction of cell viability measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. In this study cell viability after treatment with the test substance was 99.9% and 107.4% after 3 and 60 minutes, respectively. The acceptance criteria were met for the positive and negative control. Therefore, 4-(octadecylamino)-4-oxoisocrotonic acid is not considered to be corrosive to the skin.

In vitro skin irritation:

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a reconstructed human skin model according to OECD guideline 439 and in compliance with GLP (LAUS, 2017b). Skin irritation potential was identified by reduction of cell viability measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. In this study, the mean cell viability of three tissues after treatment with the test substance was 100.9%. The acceptance criteria were met for the positive and negative control. In this study the test substance did not show irritating properties towards human-derived epidermal keratinocytes. Therefore, 4-(octadecylamino)-4-oxoisocrotonic acid is not considered to be a skin irritant.

In vitro eye irritation:

The eye irritation potential of the test substance was investigated in an in vitro study using a reconstructed human cornea-like epithelium according to OECD guideline 492 and observing GLP conditions (LAUS, 2018a). The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 h. After treatment, the substance was rinsed from the tissue. Then the cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan formation was evaluated by measuring the optical density (OD) of the resulting solution. As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement. Therefore, an additional test for intensely coloured test items was performed. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the expected results. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5 (OD was 2.0). The positive control showed clear eye irritating effects as the mean value of the relative tissue viability was 38.2 % (< 50%). Variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 103.7 %. This value is well above the threshold for identifying an eye irritation potential (≤ 60%). Therefore, the test item is considered not to be irritating to eyes.

Justification for classification or non-classification

The available data on skin irritation / corrosion and eye irritation of the registered substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are, therefore, conclusive but not sufficient for classification.