Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin corrosion/irritation:

Negative for corrosion, OECD 431 (corrosion), Groot (2020)

postive for irritation (Skin Irrit. 2), OECD 439 (irritation), de Jong (2020)

Serious eye damage/irritation:

Inconclusive, OECD 437 (irritation/corrosion), Gijsbrechts (2020)

positive for irritation (Eye Irrit. 2), OECD 492 (irritation/corrosion), Groot (2020)

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:
From 30 March 2020 to 17 April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is conducted in accordance with the relevant OECD test guideline and GLP. All validity criteria were met.
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
18 June 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
EpiDerm Skin Model
Source species:
other: human-derived epidermal keratinocytes
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes, purchased or derived from tissue obtained by MatTek Corposation from accredited institutions
Source strain:
other: Keratinocyte strain 00267
Details on animal used as source of test system:
N/A
Justification for test system used:
The test is based on the experience that corrosive chemicals show cytotoxic effects following short-term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin corrosion potential of a test item by assessment of its effect on a three-dimensional human epidermis model (1-4).
The test consists of topical application of the test item on the skin tissue for 3-minute and
1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 33008 kit G+B and 33018 kit G+F
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified
- Date on Certificate of Analysis: 1 April 2020
- Date of initiation of testing: Not specified

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C ± 1.0°C
- Temperature of post-treatment incubation (if applicable): 37°C ± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) 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
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: Not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 4

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1, although initially the tissues treated for 3 minutes with the test item did not fulfill the acceptability criteria. This part of the experiment was rejected and repeated.

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%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL
Duration of treatment / exposure:
3 minutes, 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
4
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute application
Value:
74
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% tissue viability
Positive controls validity:
valid
Remarks:
8.2% tissue viability
Remarks on result:
other: no indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour application
Value:
27
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% tissue viability
Positive controls validity:
valid
Remarks:
8.8% tissue viability
Remarks on result:
other: no indication of corrosion
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: The solutions did not turn blue/purple nor a blue/purple precipitate was observed so it was concluded that the test item did not interfere with the MTT endpoint
- Colour interference with MTT: The solutions did not turn blue/purple nor a blue/purple precipitate was observed so it was concluded that the test item did not interfere with the MTT endpoint

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - 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.
- Acceptance criteria met for positive control: Yes - the mean relative tissue viability following the 1-hour exposure to the positive control was 8.8%.
- Acceptance criteria met for variability between replicate measurements: Yes - in the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was  27%, indicating that the test system functioned properly

Table 1          
Mean Absorption in the in vitro Skin Corrosion Test with N-Phenyl-diethanolamine, reaction products with formaldehyde EC: 942-131-4

 

3-minute application

1-hour application

A (OD570)

B (OD570)

Mean

(OD570)

SD

A (OD570)

B (OD570)

Mean

(OD570)

SD

Negative control

1.769

1.737

1.753

±

0.022

1.733

1.616

1.674

±

0.083

Test item

1.398

1.189

1.294

±

0.148

0.383

0.527

0.455

±

0.102

Positive control

0.155

0.134

0.144

±

0.015

0.124

0.169

0.147

±

0.032

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0436 and 0.0424 for the  3-minute and 1 hour application, respectively).

Isopropanol was used to measure the background absorption.

Table 2          
Mean Tissue Viability in the in vitro Skin Corrosion Test with N-Phenyl-diethanolamine, reaction products with formaldehyde EC: 942-131-4

 

3-minute application

viability (percentage of control)

1-hour application

viability (percentage of control)

Negative control

100

100

Test item

74

27

Positive control

8.2

8.8

 

Table 3          
Coefficient of Variation between Tissue Replicates

 

3 minute

1 hour

Negative control

1.8

6.8

Test item

15

27

Positive control

14

27

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Individual OD Measurements at 570 nm

 

3-minute application (OD570)

  A           B

1-hour application (OD570)

  A           B

Negative control

OD570 measurement 1

OD570 measurement 2

OD570 measurement 3

 

 

1.8317

1.7845

1.8072

1.6497

1.8198

1.7895

1.7597

1.6520

1.7851

1.7682

1.7583

1.6720

Test item

OD570 measurement 1

OD570 measurement 2

OD570 measurement 3

 

 

1.4371

1.2239

0.4353

0.5674

1.4380

1.2386

0.4108

0.5683

1.4499

1.2365

0.4292

0.5719

Positive control

OD570 measurement 1

OD570 measurement 2

OD570 measurement 3

 

 

0.1972

0.1786

0.1699

0.2097

0.1992

0.1761

0.1654

0.2136

0.1987

0.1771

0.1645

0.2121

OD = Optical density

Duplicate exposures are indicated by A and B.

 

Interpretation of results:
GHS criteria not met
Conclusions:
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. At the end of the treatment period. Cell viability was assessed using the MTT assay.

The positive control had a mean relative tissue viability of 8.8% 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  ≤27%, indicating that the test system functioned properly.

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 74% and 27%, 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.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 June 2020 to 05 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is conducted in accordance with the relevant OECD test guideline and GLP. All validity criteria were met.
Qualifier:
according to guideline
Guideline:
EU Method 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
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
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
EpiDerm Skin Model (EPI-SIT, Lot no.:30870 kit G)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EpiDerm test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (Epi-SIT)
- Tissue batch number(s): Lot no.: 30870
- Production date: Not stated
- Shipping date: Not stated
- Delivery date: Not stated
- Date of initiation of testing: 2nd June 2020

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Washed with Dulbecco's phosphate buffered saline (DPBS)
- Observable damage in the tissue due to washing: Not Stated
- Modifications to validated SOP: Not stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5mg/ml diluted 1:5 with MTT diluent
- Incubation time: 3hrs +/- 5 minutes
- Spectrophotometer: TECAN Infinite(R) M200 Pro Plate Reader
- Wavelength: 570nm
- Filter: Not stated
- Filter bandwidth: Not Stated
- Linear OD range of spectrophotometer: Not stated

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Not stated
- Barrier function: Not stated
- Morphology: Not stated
- Contamination: Not stated
- Reproducibility: Not stated

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues : Living epidermis is transferred to a freezer (≤-15°C), thawed and then transferred to (≤-15°C). The freeze-killed epidermis will be stored at ≤-15°C untill use.
- N. of replicates : two
- Method of calculation used: Nonspecific MTT reduction (NSMTT) will be calculated. NSMTT is the difference between the mean OD of the untreated freeze-killed tissues (ODkt_u+MTT) and test item treated freeze- killed tissues (ODkt_t+MTT) expressed as percentage of the mean of the negative control tissues (ODlt_u+MTT).
%NSMTT = [(ODkt_t+MTT – ODkt_u+MTT)/ mean ODlt_u+MTT] * 100
True tissue viability is calculated as the difference between the living test item treated tissues incubated with MTT medium (ODlt_t+MTT) and the difference between ODkt_t+MTT and ODkt_u+MTT.
OD= ODlt_t+MTT – (ODkt_t+MTT-ODkt_u+MTT)
%Viability = [OD/ mean ODlt_u+MTT] * 100
In case the %NSMTT ≤ 0.0, there is no need to correct for interference of the test item.

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

PREDICTION MODEL / DECISION CRITERIA

- The test substance is considered to be irritating to skin if the mean percent tissue viability after
exposure and post-treatment incubation is less than or equal (≤) to 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied : 30μL
- Concentration: undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): NA
- Concentration (if solution): NA
- Lot/batch no. (if required): NA
- Purity: NA

NEGATIVE CONTROL
- Amount applied : 30μL
- Concentration (if solution): Not stated

POSITIVE CONTROL
- Amount(s) applied: 30 μL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 ± 1 minutes
Duration of post-treatment incubation (if applicable):
42 hour
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
2.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
Mean tissue viability = 100%
Positive controls validity:
valid
Remarks:
Mean tissue viability = 5.5%
Remarks on result:
positive indication of irritation

Table 1: Mean absorption in the In Vitro Skin Irritation Test with N-Phenyl-diethanolamine, reaction products with formaldehyde

  A (OD570) B (OD570) C (OD570) Mean    SD
Negative contol  2.006 1.613 1.971 1.863

+/-

0.217
Test Item  0.060 0.052 0.047 0.053 

+/-

0.007
Positive Control  0.107 0.097 0.104 0.102

+/-

0.005

OD = Optical density

SD = standard deviation

Triplate exppsures are indicated by A, B and C

In this table the values are corrected for background absorption (0.0431). Isopropanol was used to measure the background absorption. 

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
The test chemical was found to be non-corrosive (Groot, 2020) according to OECD TG 431, with results from OECD TG 439 showing tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
Conclusions:
Under the conditions of the RHE test method the test substance showed irritant properties. The mean relative tissue viability was< 50% (2.8%) after 60 minute treatment with the test substance. The available data on skin irritant of the test substance does meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive and sufficient for classification.
Executive summary:

The study was performed to OECD TG 439 under GLP to assess the irritancy potential of the test material to the skin following a single application in the Epiderm (EPI-SIT) skin model. A volume 30μL of the test material undiluted was applied directly on top of the skin tissue for 60 ± 1 minutes. Assessment of skin irritation was made after a 42 hour post-incubation period. A positive and negative control were used in the study, 5% (aq) Sodium dodecyl sulfate and Dulbecco's phosphate nuffered saline respectively. A volume of 30μL of each substance was applied to the Epiderm skin model concurrently. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 60 ± 1 minutes treatment with the test item compared to the negative control tissues was 2.8%.The positive and negative control had a mean cell viability of 5.5%  and 100% respectively after 60 ± 1 minutes exposure. Since the mena relative tissue viability for the test item was below or equal to 50% after 60 ± 1 minutes treatment it is considered to be an irritant. 

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 March 2020 to 23rd March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is conducted in accordance with the relevant OECD test guideline and GLP. All validity criteria were met.
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:
09 October 2017
Deviations:
no
GLP compliance:
yes
Species:
cattle
Strain:
other: n/a
Details on test animals or tissues and environmental conditions:
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 and were tested the day of arrival in the laboratory.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL
Duration of treatment / exposure:
10 ± 1 minutes
Duration of post- treatment incubation (in vitro):
120 minutes ± 1 minutes
Number of animals or in vitro replicates:
3 replicates
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 Duratec Analysentechnik GmbH (Hockenheim, 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, Duratec GmbH). 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

NEGATIVE CONTROL USED
physiological saline

SOLVENT CONTROL USED (if applicable)
not applicable

POSITIVE CONTROL USED
ethanol

APPLICATION DOSE AND EXPOSURE TIME
750 µL, 10 ± 1 minutes

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item 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 ºC. 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.

POST-INCUBATION PERIOD: Yes. 120 ± 10 minutes at 32 ± 1°C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2 (MEM with phenol red; cMEM)
- POST-EXPOSURE INCUBATION: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated.

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 = [(Io/I)-O.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 microtiter plate reader (OD490).
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.
- Others (e.g, pertinent visual observations, histopathology): N/A

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, and UN GHS Category:
≤ 3, No Category
> 3; ≤ 55, No prediction can be made
>55, Category 1

DECISION CRITERIA: the decision criteria as indicated in the TG was used.
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
6.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
23
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - the mean negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range
- Acceptance criteria met for positive control: Yes - the mean in vitro irritancy score was 42 and within two standards deviations of the current historical positive control mean

 Table 1
Summary of Opacity, Permeability and In Vitro Scores

Treatment

Mean

Opacity 1

Mean

Permeability 1

Mean In vitroIrritation Score 1, 2

Negative control

2.6

0.019

2.9

Positive control

(Ethanol)

19

1.521

42

Test item

6.4

1.134

23

1     Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.

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

Table 2 Opacity Score

Treatment

Opacity before treatment

Opacity after treatment

Final Opacity1

Negative control corrected Final Opacity 2

Mean Final Opacity

 

 

 

 

 

 

Negative control

1.6

3.9

2.2

 

2.6

1.4

4.0

2.6

2.2

5.2

3.0

 

 

 

 

 

 

Positive control

3.4

29.0

25.6

23

19

3.6

25.3

21.8

19

1.2

20.0

18.8

16

 

 

 

 

 

 

Test item

2.2

12.3

10.0

7.4

6.4

2.4

11.4

9.0

6.4

3.0

11.2

8.2

5.6

Calculations are made without rounding off.

1    Final Opacity = Opacity after treatment – Opacity before treatment.

2    Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.

Table 3          
Permeability Score Individual Values (Uncorrected)

Treatment

Dilution factor

OD490

OD490

OD490

Average OD

Final OD

Mean final negative control

1

2

3

 

 

 

 

 

 

 

 

Negative control

1

-0.004

-0.003

-0.002

-0.003

-0.003

0.019

1

-0.004

-0.004

-0.002

-0.003

-0.003

1

0.064

0.064

0.066

0.065

0.065

 

 

 

 

 

 

 

 

Positive control

1

1.395

1.355

1.389

1.380

1.380

 

6

0.450

0.359

0.371

0.393

2.360

 

1

0.974

0.991

0.972

0.979

0.979

 

 

 

 

 

 

 

 

 

Test item

1

1.138

1.110

1.122

1.123

1.123

 

1

0.933

0.930

0.928

0.930

0.930

 

1

1.442

1.411

1.365

1.406

1.406

 

Calculations are made without rounding off.

Table 4          
Permeability Score Individual Values (Corrected)

Treatment

Dilution factor

Negative control corrected OD490 11

Negative control corrected OD490 21

Negative control corrected OD490 31

Negative control corrected OD490

Average

Negative control corrected final

OD490

Average OD

 

 

 

 

 

 

 

 

Positive control

1

1.376

1.336

1.370

1.360

1.360

1.521

6

0.431

0.340

0.352

0.374

2.243

1

0.955

0.972

0.953

0.960

0.960

 

 

 

 

 

 

 

 

Test item

1

1.119

1.091

1.103

1.104

1.104

1.134

1

0.914

0.911

0.909

0.911

0.911

1

1.423

1.392

1.346

1.387

1.387

Calculations are made without rounding off.

1    OD490 values corrected for the mean final negative control permeability (0.019).

 

Table 5          
In Vitro Irritancy Score

Treatment

Final Opacity2

Final OD4902

In vitro Irritancy Score 1

 

 

 

 

Negative control

2.2

-0.003

2.2

2.6

-0.003

2.6

3.0

0.065

4.0

 

 

 

 

Positive control

23

1.360

43

19

2.243

53

16

0.960

31

 

 

 

 

Test item

7.4

1.104

24

6.4

0.911

20

5.6

1.387

26

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

2   Positive control and test item are corrected for the negative control.

 

 

 

 

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the condition of this study, the test item induced an IVIS > 3 ≤ 55 and it was concluded that no prediction on the classification can be made in respect to its potential to cause eye irritation.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item, 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 study 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 test item was applied undiluted (750 μL) directly on top of the corneas.

The mean 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 42 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 induced a mean in vitro irritancy score of 23 after 10 minutes of treatment which falls within the IVIS > 3 ≤ 55 category.

Under the condition of this study, the test item induced an IVIS > 3 ≤ 55 and it was concluded that no prediction on the classification can be made in respect to its potential to cause eye irritation.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March 2020 - 10 April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is conducted in accordance with the relevant OECD test guideline and GLP. All validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
18 June 2019
GLP compliance:
yes (incl. QA statement)
Species:
human
Strain:
other: Normal human keratinocytes, strain 4F1188
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
Rationale for test method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
EpiOcular(TM) (OCL-200-EIT MatTek Corporation, Ashland, MA; Lot: 31733 kit F; Part No. OCL-200, OCL-212)
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The liquid test item was applied undiluted (50 μL) directly on top of the tissue.
- Concentration (if solution): N/A

VEHICLE
N/A
Duration of treatment / exposure:
30 minutes ± 2 minutes
Observation period (in vivo):
N/A
Duration of post- treatment incubation (in vitro):
2 hours ± 15 minutes
Number of animals or in vitro replicates:
6 well-plates
Details on study design:
- Details of the test procedure used:
See 'Any other details on materials and methods'

- RhCE tissue construct used, including batch number: OCL-200-EIT, Lot: 31733 kit F
- Doses of test chemical and control substances used:
Test item: 50 μL
Postive control: 50 μL
Negative control: 50 μL

- Duration and temperature of:
Exposure: 30 ± 2 minutes at 37.0 ± 1.0°C
Post-exposure immersion: 12 ± 2 minute immersion incubation at room temperature
Post-exposure incubation: 120 ± 15 minutes at 37°C

- Justification for the use of a different negative control than ultrapure H2O (if applicable): N/A
- Justification for the use of a different positive control than neat methyl acetate (if applicable): N/A
- Description of any modifications to the test procedure: N/A
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): A negative control, 50 μL sterile Milli-Q water was tested concurrently.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
Test item, postive and negative control: 2 tissues
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): Extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Description of the method used to quantify MTT formazan:
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. Formazan was extracted with 2 mL isopropanol refrigerated for 18 ± 2 hours in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken. Extracted formazan was determined spectrophotometrically. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not reported
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.

The test chemical is identified as “no prediction can be made” if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The positive control had a mean cell viability after 30 ± 2 minutes exposure of 28%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

- Complete supporting information for the specific RhCE tissue construct used: N/A

- Reference to historical data of the RhCE tissue construct: Not reported

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Not reported

- Positive and negative control means and acceptance ranges based on historical data:
Range Mean
Negative control (absorption; OD570) 1.077-2.070 1.711
Positive control (absorption; OD570) 0.029 – 0.823 0.413
Positive control (viability; %) 2.11 – 45.20 23.95

- Acceptable variability between tissue replicates for positive and negative controls: The difference between the percentage of viability of two tissues treated identically was less than 2%, indicating that the test system functioned properly. Historical control data were obtained between December 2016 and December 2019.
- Acceptable variability between tissue replicates for the test chemical: Yes
Irritation parameter:
other: mean tissue viability
Run / experiment:
Mean
Value:
4.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The test item was checked for possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed it was concluded that the test item interacted with the MTT endpoint.
In addition, the test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0007 and 0.0012, respectively. Therefore, it was concluded that the test item did not induce color interference.
Since the test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was -0.16% of the negative control tissues. Since the %NSMTT ≤ 0.0, there is no need to correct for interference of the test item.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline:

Range Mean SD n
Negative control (absorption; OD570) 1.077-2.070 1.711 0.231 48
Positive control (absorption; OD570) 0.029 – 0.823 0.413 0.217 48
Positive control (viability; %) 2.11 – 45.20 23.95 11.92 48

The mean absorption at 570 nm measured after treatment with the test item and controls are presented in Table 1. The values are corrected for background absorption (0.0432). Isopropanol was used to measure the background absorption.

Table 2 shows the mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 4.7 %. Since the mean relative tissue viability for the test item was below 60 % it is considered to be potentially irritant or corrosive to the eye.

The positive control had a mean cell viability after 30 ± 2 minutes exposure of 28 %. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 2 %, indicating that the test system functioned properly.

Table 1. Mean Absorption in the EpiOcular Test with the test item

 

A

(OD570)

B

(OD570)

Mean

(OD570)

SD

Negative control

1.847

1.842

1.845

±0.004

Test item

0.077

0.097

0.087

± 0.014

Positive control

0.518

0.504

0.511

± 0.010

OD = optical density   SD = Standard deviation

Duplicate exposures are indicated by A and B.

 

Table 2. Mean Tissue Viability in the EpiOcular Test with the test item

 

Mean tissue viability (% of control)

Difference between two tissues (%)

Negative control

100

0.29

Test item

4.7

1.04

Positive control

28

0.73

 

Table 3. Individual optical density measurements at 570 Nm

 

A

(OD570)

B

(OD570)

Negative control

OD570 measurement 1

OD570 measurement 2  

 

1.8738

1.9074  

 

1.8890

1.8815

Test item

OD570 measurement 1

OD570 measurement 2  

 

0.1195

0.1217

 

0.1402

0.1392

Positive control

OD570 measurement 1

OD570 measurement 2   

 

0.5623

0.5592

 

0.5486

0.5458

OD = optical density

Duplicate exposures are indicated by A and B.

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Executive summary:

A test was performed in accordance with OECD 492 (2019), in order to determine the eye irritation potential of the test item. The test item was topically applied, undiluted at a dose of 50 µL directly on top of the tissue of the Reconstructed Human EpiOcular Model for 30 ± 2 minutes.

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

Positive and negative controls were run concurrently. The positive control had a mean cell viability of 28 % after 30 ± 2 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 difference between the percentage of viability of two tissues treated identically was less than 2 %, indicating that the test system functioned properly.

The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was -0.16 % of the negative control tissues. The ODs of the test item treated viable tissues was corrected using the OD of the freeze-killed tissues.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 4.7 %. Since the mean relative tissue viability for the test item was below or equal to 60 % after 30 ± 2 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

In conclusion, the test item is considered to be potentially irritant or corrosive to the eye. No specific prediction can be made regarding the classification based on the EpiOcular test results. Since it is not possible to conclude whether the test item is an Eye Irritant Category 2A or 2B, the classification is concluded to be: Eye Irritant Category 2 in accordance with CLP Regulation No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion/irritation

A skin corrosion study was performed in accordance with OECD 431 to determine the potential corrosive properties of the test substance. Due to the negative outcome of this study, a skin irritation study was performed in accordance with OECD 439 to determine the skin irritation potential of the test substance.

 

OECD 431

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 test item was applied undiluted (50 µL) directly on top of the skin tissue. At the end of the treatment period cell viability was assessed using the MTT assay.

The positive control and negative control tissues were valid. The Coefficient of Variation were also valid.

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 74% and 27%, 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.

 

OECD 439

The objective of this study was to evaluate the test item, N-Phenyl-diethanolamine, reaction products with formaldehyde EC: 942-131-4 for its ability to induce skin irritation on a human three dimensional epidermal model (EpiDerm(Epi-SIT)). The possible irritant potential of the test item was tested through topical application for 60 ± 1 minutes with a 42 hour post incubation period. The test item was applied undiluted (30 µL) directly on top of the skin tissue.

The positive and negative control tissues were valid. The absolute mean OD570 (optical density at 570nm) if the negative control tissues were within the laboratory historical control data range and within the acceptance limits of OECD439 . The standard deviation value of percentage viability indicated the test system functioned properly confirming the testing system and the quality of the tissues.

Skin irritation is expressed  as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 60 ± 1 minute’s treatment with the test item compared to the negative control tissues  was 2.8%. Since the mean relative tissue viability for the test item was below or equal to 50% after 60 ± 1 minute’s treatment it is considered to be an irritant.

In conclusion, the test item is a skin irritant in the in vitro skin corrosion test under the experimental conditions described in this report.

 

Serious eye damage/irritation

A study was performed in accordance with OECD 437 to determine the potential for serious eye damage or eye irritant properties of the test substance. As a classification prediction could not be made based on the OECD 437, an additional study was performed in accordance with OECD 492 to enable a conclusion on classification for this endpoint.

Considering results from both the OECD 437 and the OECD 492, a classification of Eye Irritation Category 2 in concluded in accordance with CLP Regulation No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.

 

OECD 437

The objective of this study was to evaluate the eye hazard potential of the test item, 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 study 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 test item was applied undiluted (750 μL) directly on top of the corneas.

The mean negative control and positive control were valid.

The test item induced a mean in vitro irritancy score of 23 after 10 minutes of treatment which falls within the IVIS > 3 ≤ 55 category.

Under the condition of this study, the test item induced an IVIS > 3 ≤ 55 and it was concluded that no prediction on the classification can be made in respect to its potential to cause eye irritation.

 

OECD 492

A test was performed in accordance with OECD 492 (2019), in order to determine the eye irritation potential of the test item. The test item was topically applied, undiluted at a dose of 50 µL directly on top of the tissue of the Reconstructed Human EpiOcular Model for 30 ± 2 minutes.

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

Positive and negative controls were run concurrently and were valid.

The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was -0.16 % of the negative control tissues. The ODs of the test item treated viable tissues was corrected using the OD of the freeze-killed tissues.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 4.7 %. Since the mean relative tissue viability for the test item was below or equal to 60 % after 30 ± 2 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

In conclusion, the test item is considered to be potentially irritant or corrosive to the eye. No specific prediction can be made regarding the classification based on the EpiOcular test results. Since it is not possible to conclude whether the test item is an Eye Irritant Category 2A or 2B, the classification is concluded to be: Eye Irritant Category 2 in accordance with CLP Regulation No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.

Justification for classification or non-classification

Skin irritation: Based on the available in vitro studies, Skin Irritant Category 2 is applied to the test item in accordance with CLP Regulation No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.

Eye Irritation: Based on the available in vitro studies, Eye Irritant Category 2 is applied to the test item in accordance with CLP Regulation No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.