Registration Dossier

Administrative data

Description of key information

Skin irritation:

- Episkin and Epiderm: not irritating (OECD 439, GLP, K, rel. 1)

Eye irritation:

- ICE: no conclusion can be made (OECD 438, GLP, K, rel.1).

- EpiOcular: not irritating (OECD 492, GLP, K, rel.1).

Conclusion: based on the available information, the substance is not classified for eye irritation.

Respiratory irritation:

No data was available.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 July to 29 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 439 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz (inspected on July 13-16, 2015 / signed on September 14, 2015)
Test system:
human skin model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes
Justification for test system used:
Following the REACH top-down strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France).
- Tissue batch number(s): 17-EKIN-030
- Expiry date: 31 July 2017
- Date of initiation of testing: 04 July 2017
The EpiSkin™ tissue consists of NHEK, 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 EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 25 July 2017. EpiSkin™ tissues were transferred to 12-well plates with maintenance medium and the pre-incubation phase of the EpiSkin™ tissues started.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 12-well plate. The tissues were be gently rinsed with PBS to remove any residual test material. Excess PBS were removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. Tissues were incubated for 42 ± 1 hour at 37 ± 1.5 °C, 5 ± 0.5% CO2.Number of washing steps not reported.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none

IL-1 ALPHA IMMUNOASSAY
If the results obtained by means of the MTT assay are unclear or borderline, additionally the IL-1 α concentration in the medium after 42 hours incubation can be determined by order of the Sponsor. For this purpose, samples of all treatment groups are taken from the wells. The plates are shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well is taken and is stored in the freezer at ≤ -18 °C until analysis.
Following the instruction from the QuantikineTM kit the amount of released IL-1 α will be determined. Therefore 200 μL of each sample and the standard solutions is mixed with 50 μL Assay Diluent RD1C in the wells of the IL-1 α Microplate. After 2 hours incubation and washing steps 200 μL IL-1 α Conjugate is added to each well. Meanwhile the substrate solution is prepared by mixing Colour Reagent A and B in equal volumes. After 1 hour incubation and washing steps 200 μL of the substrate solution is added to each well. The plates are covered with the adhesive strips and incubated for further 20 min under light protection. After 20 min 50 μL of the stop solution is added to each well. The plates are read at 450 nm in a photometer (Versamax® Molecular Devices, software version 4.7.1) within 30 minutes. Each sample is tested in duplicate.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, software version 4.7.1
- Wavelength: 570 nm
- Filter: with 570 nm filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: 2.1 mg/L (1.5 mg/L < IC50 < 3;0 mg/L)
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: on blood of the same donors: absence of HIV1 and 2 antibodies, of hepatitis C antibodies, and hepatitis B antigen HBs. On epidermal cells of the same donors: absence of bacteria, fungus and mycoplasma.
- Reproducibility: the results for the positive and negative controls are within the historical ranges obtained by Envigo CRS GmbH in the previous eleven months (means. rel. standard deviation. and ranges) of Envigo CRS GmbH

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
Not needed

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 relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.
- For the current test, an irritation potential of a test item is predicted if the release of IL-1 α is above the threshold of 60 pg/mL.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): approximately 10 μL (26.3 μL/cm² according to guideline)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL (in DPBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Triplicate tissues for test substance, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
52.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6.9%
Remarks on result:
no indication of irritation
Remarks:
> 50 %
Irritation / corrosion parameter:
other: IL-alpha concentration (pg/L)
Run / experiment:
1
Value:
26.15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
3.14
Positive controls validity:
valid
Remarks:
129.21
Remarks on result:
no indication of irritation
Remarks:
< 50 pg/mL
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.672 and the standard deviation value of the viability was 2.8%.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.9% relative to the negative control treated tissues and the standard deviation value of the viability was 11.9%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.6%. - Reference to historical values: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

Table 7.3.1/1: Results of the MTT assay

Test Group

Absor-bance 570 nm
Tissue Well 1

Absor-bance 570 nm
Tissue Well 2

Mean Absor-bance 570 nm

Mean Absor-bance 570 nm
blank corrected

Mean Absor-bance of 3 Tissues

Relative Viability [%] Tissue
1, 2, 3*

Relative Standard Deviation [%]

Mean Rel. viability

[%]**

Blank

0.038

0.039

0.038

 

 

Negative Control

0.718

0.684

0.701

0.663

0.672

 

98.7

2.8

 

100.0

0.759

0.705

0.732

0.694

103.2

0.717

0.677

0.697

0.659

98.1

Positive Control

0.089

0.087

0.088

0.050

0.047

 

7.4

11.9

 

6.9

0.086

0.090

0.088

0.050

7.4

0.075

0.082

0.078

0.040

6.0

Test Item

0.406

0.406

0.406

0.368

0.351

54.8

5.6

52.2

0.395

0.392

0.394

0.355

52.9

0.376

0.360

0.368

0.329

49.0

* Relative absorbance [rounded values]

**  Mean relative absorbance [rounded values]

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 52.2% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

This value is very close to the threshold for irritancy (≤ 50%), consequently, the results of the MTT test were considered borderline. It was considered necessary to perform IL-1α analysis.

Table 7.3.1/2: Results of the IL1 ELISA

Abs. 1
(OD)

Abs. 2
(OD)

Mean
Abs. 1-2 (OD)

IL1αConc.
[pg/ml]*

Medium

0.020

0.017

0.019

0.903

42h Negative Ctrl, Tissue 1

0.060

0.078

0.069

4.507

42h Negative Ctrl, Tissue 2

0.059

0.055

0.057

3.641

42h Negative Ctrl, Tissue 3

0.105

0.115

0.110

7.510

42h Positive Ctrl, Tissue 1

1.447

1.554

1.501

150.458

42h Positive Ctrl, Tissue 2

1.051

1.360

1.206

113.458

42h Positive Ctrl, Tissue 3

1.189

1.482

1.336

129.320

42h Test Item, Tissue 1

0.264

0.236

0.250

18.287

42h Test Item, Tissue 2

0.503

0.451

0.477

37.482

42h Test Item, Tissue 3

0.406

0.270

0.338

25.476

*the parallel entrainment of a definite IL1α standard led to a curve with the following formula:

y = 20,654x2+ 69,547x - 0,3903


The IL1α concentration of the samples (y) was calculated by setting the value
Mean Abs. 1-2 (OD) as x.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 570 nm.

 

The relative mean viability of the test item treated tissues was 52.2% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period. The amount of mean IL1 -alpha release is 26.15 pg/mL.

The relative mean tissue viability for the positive control treated tissues was 6.9% relative to the negative control treated tissues and the standard deviation value of the viability was 11.9%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.672 and the standard deviation value of the viability was 2.8%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.6%. The test item acceptance criterion was therefore satisfied.

All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

Under the experimental conditions of this study, the viability being above 50% and the IL1 -alpha release below 50 pg/mL, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 March to 20 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 439 without any deviation.
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:
06 July 2012
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz (inspected on July 13-16, 2015 / signed on September 14, 2015)
Test system:
human skin model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200- SIT Kit
- Tissue batch number(s): 25896
- Epi-200 SIT kits and MTT-100 assay kit were purchased from MatTek Corporation (82105 Bratislava, Slovakia).
- The EpiDerm™ tissue 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) are cultured on specially prepared cell culture inserts.
- EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 17 April, 2018. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

PRE-EXPERIMENT
Test for direct MTT reduction and colour interference
- 30 μL of the test item were added to 0.3 mL of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.
Since the test item did not change colour in the presence of water, an additional test (step 2) with viable tissues (but without MTT addition) was not necessary to be performed.
- The test item was further evaluated for its potential to interfere with MTT assay. To test if a test item directly reduces MTT, 30 μL of the test item were added to 1 mL of the MTT-solution (1 mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.
Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

MAIN EXPERIMENT
Pre-warming of EpiDerm™ Tissues: The 24-well plate with epidermal tissues was opened under sterile conditions and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1. air bubbles between agarose and insert were not > 30% of the total surface,
2. liquid on top of the insert was removed with sterile cotton tips,
3. if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.
0.9 mL of the assay medium (20 – 25 °C) were pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 22.5 hours (37 ± 1.5 °C, 5 ± 0.5% CO2).

TREATMENT: After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative control, the positive control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The exposure of the tissues to the test and control items was performed at 37 ± 1.5 °C, 5 ± 0.5% CO2.
- Temperature of post-treatment incubation: Tissues were incubated at 37 ± 1.5 °C, 5 ± 0.5% CO2 for 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for 24 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. After incubation medium was changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another about 18 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. The complete incubation time was 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- 300 μL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until further use.
- Incubation time: After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates and incubated for 3 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2
- After a 3-hour incubation period, the tissues were rinsed three times with DPBS, and carefully dried with blotting paper. The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues are completely covered. The 24-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for about 2 hours while shaking at room temperature.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1) with a 570 nm filter. Mean values were calculated from the 3 wells per tissue.

NUMBER OF REPLICATE TISSUES: Triplicate tissues for test item, negative and positive controls

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Positive Control; OD at 570 nm after exposition to 5% SDS solution in deionised water (MatTek):
Mean Viability: 4.28%, Standard Deviation: 1.00, Rel. Standard Deviation: 23.44%, Range of Viabilities: 2.24%—6.19%, Mean Absorption: 0.07, Standard Deviation: 0.02, Rel. Standard Deviation: 25.96%, Range of Absorbance: 0.03—0.11
Negative Control OD at 570 nm DPBS (MatTek):
Mean Absorption: 1.66%, Standard Deviation: 0.20, Rel. Standard Deviation: 11.96%, Range of Absorbance*: 1.28—2.00

PREDICTION MODEL / DECISION CRITERIA
- Classification of irritation potential is based on relative viability according to the following:
Relative mean tissue viability is ≤50%: Irritant (Category 1 (H314) or Category 2 (H315))
Relative mean tissue viability is >50%: Non-Irritant (Not classified or UN GHS Category 3 cannot be determined)
*In cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5%, a second run should be considered, as well as a third one in case of discordant results between the first two runs.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the undiluted test item were dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue for a complete treatment time of 60 minutes.
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL DPBS was applied to triplicate tissue for 60 minutes

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of 5% SDS solution in deionised water was applied to triplicate tissue for 60 minutes
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 60 minutes.
Duration of post-treatment incubation (if applicable):
At the end of the exposure period, tissues were rinsed and incubated at 37 ± 1.5 °C, 5 ± 0.5% CO2 for 42 h.
Number of replicates:
Triplicate tissues each for test item, negative and positive controls.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure period and 42 h post-exposure incubation period
Value:
94.3
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:
PRE-EXPERIMENT:
Assessment of Color Interference with the MTT endpoint: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour after 1 hour incubation.
Direct MTT Reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue/purple colour.
The test item passed the MTT- and the Colour Interference pre-tests.

MAIN EXPERIMENT
The mean relative viability of the test item, corresponding to the cell viability, decreased to 94.3% (threshold for irritancy: ≤ 50%), consequently the test item was non-irritant to skin.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.0% thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The standard deviations between the three tissue percentage viability values of group (test item, positive and negative controls) in the main test were below 18 (threshold according OECD 439: ≤ 18), thus ensuring the validity of the study.

Table 7.3.1/1: Main test

 

Treatment Group

 

Tissue No.

 

OD 570 nm Well 1

 

OD 570 nm Well 2

 

OD 570 nm Well 3

 

Mean OD of 3 Wells

 

Mean OD of 3 Wells blank corrected

Mean OD of 3 tissues blank corrected

Rel. Viability [%] Tissue 1, 2 + 3*

 

 

Standard Deviation

 

Mean Rel. Viability [%]**

Blank

-

0.037

0.038

0.036

0.037

-

Negative Control

1

1.802

1.775

1.752

1.776

1.739

1.616

107.6

11.0

100.0

2

1.729

1.749

1.723

1.734

1.697

105.0

3

1.453

1.427

1.470

1.450

1.413

87.4

Positive Control

1

0.089

0.087

0.089

0.088

0.051

0.048

3.2

0.2

3.0

2

0.082

0.084

0.080

0.082

0.045

2.8

3

0.084

0.085

0.085

0.085

0.048

2.9

Test Item

1

1.643

1.620

1.608

1.624

1.587

1.525

98.2

6.9

94.3

2

1.660

1.594

1.632

1.629

1.592

98.5

3

1.466

1.411

1.420

1.432

1.395

86.3

 

*Relative viability [rounded values]: [(absorbanceTI/PC/NC) / (Mean absorbanceNC)] x 100

** Mean relative viability [rounded values]: [(Mean absorbanceTI/PC/NC) / (Mean absorbanceNC)] x 100

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions, test item is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EpiDermTM reconstructed human epidermis model.

 

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water (pre-test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

 

Triplicate tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (PBS) or the positive control (5% SDS) for 60 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post exposure incubation period each tissue was taken for MTT-loading. A total biopsy of each epidermis was made and placed into 24-well plates containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre labeled 96 well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

 

After treatment with the test item, the mean relative viability value decreased to 94.3% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

 

Under the experimental conditions, test item is not irritant to skin according to UN GHS and EU CLP regulation.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 438 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
Adopted 26 July 2013
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Multiple chicken heads were supplied by XXX.
- Characteristics of donor animals (e.g. age, sex, weight): Spring chickens (Gallus Gallus e.g. Ross 308 Broiler) male or female, weighed approximately 3kg and were approximately 56 days old.
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized. All eyes fall within the acceptance criteria identified in the test guideline. The temperature of the chambers was at 32 ±1.5 °C.
Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
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): 30 µL of the test item was applied, as supplied, to the cornea
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
The test item was applied for 10 seconds and then rinsed from the eye using 20mL of 0.9% (w/v) sodium chloride solution (pre-warmed to approximately 32 °C).
Number of animals or in vitro replicates:
Test item: 3 eyes
Positive control: 3 eyes
Negative control: 2 eyes
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are =< 0.5.
- Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
- Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.
- Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit lamp microscope at the center of each cornea.
- Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.
- After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES: 2, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: sodium chloride 0.9% w/v

POSITIVE CONTROL USED: 5% Benzalkonium chloride

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

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

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. 1.
- Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
- Mean maximum opacity score: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein
- Pitting, loosening (sloughing), roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

DECISION CRITERIA:
Results from corneal opacity, swelling, and fluorescein retention should be evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
>= 0.5 - <= 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
1
Value:
>= 3.74 - <= 13.08
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 2.0 (180-240 min.), corresponding to the ICE class III;
- mean score of fluorescein retention: 0.7 corresponding to the ICE class II;
- maximal mean corneal swelling: 13.08% (75 min.) corresponding to the ICE class III.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

HISTORICAL CONTROL DATA:
In order to confirm the acceptability of the test, a comparison was made with historical control data for negative and positive controls obtained by the laboratory. The test was considered acceptable as the concurrent negative and positive controls were identified as GHS Non-Classified and GHS Category 1 respectively.

Table 7.3.2/5: Individual scores and Mean scores for corneal effects - test item

 

End Point

Eye Number

Time
(after decontamination)

0
minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

3A

0.5

0.5

1

2

2

2

6A

0.5

0.5

2

2

2

2

8A

0.5

0.5

0.5

1

2

2

Mean

0.5

0.5

1.2

1.7

2.0

2.0

ICE Class

III

Fluorescein Retention

3A

 

1

 

 

 

 

6A

 

0.5

 

 

 

 

8A

 

0.5

 

 

 

 

Mean

 

0.7

 

 

 

 

ICE Class

II

Corneal Thickness

3A

0.72

0.70

0.84

0.77

0.78

0.76

6A

0.72

0.78

0.80

0.78

0.76

0.76

8A

0.70

0.76

0.78

0.84

0.74

0.70

Mean

0.71

0.75

0.81

0.80

0.76

0.74

Mean Corneal Swelling (%)

 

4.67

13.08

11.68

6.54

3.74

ICE Class

III

Corneal Epithelium Condition

3A

N

N

N

N

N

N

6A

N

N

N

N

N

N

8A

N

N

N

N

N

N

ICE Classes Combined:

1 x II, 2 x III

Classification:

No prediction can be made

Table 7.3.2/6: Individual scores and Mean scores for Corneal Effects - positive control

End Point

Eye Number

Time
(after decontamination)

0
minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

2A

0.5

3

3

4

4

4

5A

0.5

3

4

4

4

4

7A

0.5

4

4

4

4

4

Mean

0.5

3.3

3.7

4.0

4.0

4.0

ICE Class

IV

Fluorescein Retention

2A

 

3

 

 

 

 

5A

 

3

 

 

 

 

7A

 

3

 

 

 

 

Mean

 

3.0

 

 

 

 

ICE Class

IV

Corneal Thickness

2A

0.68

0.80

0.88

0.84

0.80

0.82

5A

0.72

0.83

0.88

0.92

0.94

0.94

7A

0.72

0.90

0.90

0.97

0.90

0.92

Mean

0.71

0.84

0.89

0.91

0.88

0.89

Mean Corneal Swelling (%)

 

19.34

25.47

28.77

24.53

26.42

ICE Class

III

Corneal Epithelium Condition

2A

N

N

N

N

N

N

5A

N

S

S

S

S

S

7A

N

N

N

N

N

N

ICE Classes Combined:

2 x IV, 1 x III (sloughing in 1 eye)

Classification:

Category 1

Table 7.3.2/7: Individual scores and Mean score for Corneal effects - Negative controlthe test item

End Point

Eye Number

Time
(after decontamination)

0
minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

1A

0.5

0.5

0.5

0.5

0.5

0.5

4A

0.5

0.5

0.5

0.5

0.5

0.5

Mean

0.5

0.5

0.5

0.5

0.5

0.5

ICE Class

I

Fluorescein Retention

1A

 

0.5

 

 

 

 

4A

 

0.5

 

 

 

 

Mean

 

0.5

 

 

 

 

ICE Class

I

Corneal Thickness

1A

0.68

0.70

0.70

0.68

0.70

0.70

4A

0.72

0.71

0.74

0.72

0.70

0.72

Mean

0.70

0.71

0.72

0.70

0.70

0.71

Mean Corneal Swelling (%)

 

0.71

2.86

0.00

0.00

1.43

ICE Class

I

Corneal Epithelium Condition

1A

N

N

N

N

N

N

4A

N

N

N

N

N

N

ICE Classes Combined:

3 x I

Classification:

No Category

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the test conditions, no prediction can be made for the test substance according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

The test item was applied, as supplied, at the dose of 30 µL, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed using 20 mL of isotonic saline. Three eyes were treated in the same manner with a positive control and two eyes with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 2.0 (180-240 min.), corresponding to the ICE class III;

- mean score of fluorescein retention: 0.7 corresponding to the ICE class II;

- maximal mean corneal swelling: 13.08% (75 min.) corresponding to the ICE class III.

 

The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

 

Under the test conditions, no prediction can be made for the test substance according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for in vitro eye irritation endpoint.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 February to 01 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 492 without any deviation.
Qualifier:
according to
Guideline:
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)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz (inspected on July 13-16, 2015 / signed on September 14, 2015)
Species:
human
Details on test animals or tissues and environmental conditions:
EpiOcular Kit Components Needed for the Assay
Lot No.: 27025
Sealed 24-well plate: Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium: DMEM-Medium
12-well plate: Holding plate
24-well plates: For MTT viability assay
6-well plates: For storing inserts, or for topically applying test agents
Ca++Mg++-Free DPBS: Dulbecco´s Phosphate Buffered Saline

EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts. EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (27 February 2018) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates. Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) overnight (about 17 hours).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL of the test item were dispensed directly atop duplicate EpiOcular™ tissue for 30 minutes.
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
30 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2
Duration of post- treatment incubation (in vitro):
120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation)
Number of animals or in vitro replicates:
2 tissue replicates each for test item, negative and positive controls
Details on study design:
PRE-EXPERIMENT
Assessment of direct MTT reduction by the test item: 50 μl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. If the MTT solution colour turned blue/purple, the test item is presumed to have reduced the MTT.
Since the MTT solution colour did not turn blue/purple, the test item is not presumed to be a MTT reducer. An additional test with freeze-killed tissues did not have to be performed.
Assessment of coloured or staining materials: 50 μL each of the test item were added either to 1.0 mL of water or to 2 mL isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour, the isopropanol mixture for 3 hours at room temperature.
Since the test item did not dye water or isopropanol, additional tests with viable tissues did not have to be performed.

- Doses of test chemical and control substances used : 50 μL

Experimental Performance
- After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 μL topically on the EpiOcular™ tissues. The tissues were incubated for 30 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 .
At the end of the 30 minutes treatment time, each tissue was rinsed with Ca++Mg++-free DPBS, incubated for 12 minutes at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

- Number of tissue replicates used per test chemical and controls (positive control, negative control) : 2 tissue replicates each for test item, negative and positive controls

- MTT Assay: At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
If the test item-treated tissue viability is > 60% relative to the negative control-treated tissue viability, the test item is labeled non-irritant (no Category according to UN GHS).
If the test item-treated tissue viability is ≤ 60% relative to negative control-treated tissue viability, the test item is labeled irritant (Category 2 or Category 1 according to UN GHS; no differentiation between the categories possible).

- Positive and negative control means and acceptance ranges based on historical data
Positive Control:
Mean Viability: 32.3%, Rel. Standard Deviation: 11.1%, Range of Viabilities: 6.90% - 43.4%
Mean Absorption: 0.566, Rel. Standard Deviation: 0.283, Range of Absorbance: 0.107- 0.943
Negative Control:
Mean Absorption: 1.65, Rel. Standard Deviation: 0.295, Range of Absorbance: 1.27 – 2.16

- Acceptability of the Assay
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.
The positive and negative control data shall fall within the historical control data.
Irritation parameter:
other: relative mean viability of the tissues (%)
Run / experiment:
30 minutes
Value:
100.7
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:
PRE-EXPERIMENT
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue/purple colour.
Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured intensively, did not dye water or isopropanol, and did not prove to be a MTT reducer.

MAIN EXPERIMENT
- Irritating effects were not observed following incubation with test item.
- The mean relative absorbance value of the test item, corresponding to the cell viability, was 100.7% (threshold for irritancy: ≤ 60%), consequently the test item was not irritant to eye.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD is > 0.8 and < 2.5 (1.956 and 2.064)
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (41.2%). Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 41.2%, thus the validity of the test system is ensured.
- The difference of viability between the two replicates is < 20% (values between 0.8% and 2.6%) in the same run (for positive and negative control tissues and tissues of single test items).

Table 7.3.2/1: Main test

 

Treatment Group

 

Tissue No.

 

OD 570 nm Well 1

 

OD 570 nm Well 2

 

Mean OD of 2 Wells

 

Mean OD of 2 Wells blank

corrected

Mean OD of Treatment Group

blank corrected

Rel. Viability [%] Tissue 1, 2 *

 

Absolute Value of the Difference of Rel. Viability Tissue 1,2 [%]

 

Mean Rel. Viability

[%]**

Blank

 -

0.038

0.038

0.038

0.000

Negative Control

1

2.064

1.989

2.027

1.988

1.963

101.3

2.6

100.0

2

1.995

1.956

1.975

1.937

98.7

Positive Control

1

0.871

0.840

0.856

0.817

0.809

41.6

0.8

41.2

2

0.851

0.828

0.840

0.801

40.8

Test Item

1

2.076

1.978

2.027

1.989

1.977

101.3

1.2

100.7

2

2.006

2.000

2.003

1.965

100.1

*Relative viability [rounded values]: [(absorbanceTI/PC/NC) / (Mean absorbanceNC)] x 100

** Mean relative viability [rounded values]: [(Mean absorbanceTI/PC/NC) / (Mean absorbanceNC)] x 100

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions, test item does not possess any eye irritating potential (no Category according to UN GHS) since the mean percent tissues viability after exposure and post-exposure incubation is higher than 60%.
Executive summary:

An in vitro study was performed to assess the eye irritation potential of test item by means of the Human Cornea Model Test (EpiOcularTM model) according to the OECD Guideline 492 and in compliance with GLP.

 

The test item did not prove to be a MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

 

50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes. At the end of the treatment period, each tissue was rinsed with Ca++Mg++-free DPBS, incubated for 12 minutes at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation). The cell viability was then assessed by means of the colorimetric MTT reduction assay. Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability (41.2%) compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

 

Irritating effects were not observed following incubation with test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (determined value for the test item: 100.7%).

 

Under the experimental conditions, test item does not possess any eye irritating potential (no Category according to UN GHS) since the mean percent tissues viability after exposure and post-exposure incubation is higher than 60%.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion:

Since no key study was identified on the substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the skin corrosion/irritation potential of the substance:

 

Element

Information

Conclusion

Comments

Existing data on physico
- chemical properties

1a

Is the substance spontaneously flammable in contact with air (pyrophoric) or water at room temperature?

NO

 

1b

Is the substance an organic hydroperoxide or an organic peroxide?

NO

 

1c

Is the pH of the substance ≤ 2.0 or ≥ 11.5?

NO

 

1d

Are there other physical or chemical properties that
indicate that the substance is corrosive/irritant?

NO

 

Existing human data

2

Are there adequate existing human data which provide evidence that the substance is a corrosive
or irritant?

NO

 

Existing animal data from corrosion/irritation studies

3

Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?

NO

 

Existing data from general toxicity studies via the dermal route and from sensitisation studies

4a

Is the substance classified as fatal in contact with skin (LD50 ≤ 50 mg/kg bw, CLP hazard statement
H310)

NO

Dermal LD50 > 5000 mg/kg bw

4b

Has the substance proven to be a corrosive, irritant or non-irritant in a suitable acute dermal toxicity test?

NO

The acute dermal toxicity test was performed on abraded skin only, therefore results were not considered suitable.

4c

Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated
exposure?

NO

LLNA available: tested only up to 50%. No sign of irritation or corrosion leading to C&L.
NB: In general, irritation data from the Local Lymph Node Assay are not usable. The test substance is applied to the dorsum of the ear by open topical application

Existing/new (Q)SAR data and read
-across

5a

Are there structurally related substances (suitable “read-across” or grouping), which are classified as corrosive to the skin (Skin Corrosive Cat. 1), or do suitable (Q)SAR methods indicate corrosion
potential of the substance?

NO

Predicted to be "Not irritating to Skin" using Toxtree (v2.6.6)
OASIS TIMES (v2.27.17) did not indicate any skin irritation alert but the prediction was not supported by sufficient applicability domain.

5b

Are there structurally related substances (suitable “read-across” or grouping), which are classified as irritant to the skin (Skin Irritant Cat. 2), or indicating that the substance is non-irritant, or do suitable (Q)SAR methods indicate irritant or non-irritant potential of the substance?

NO

 

Existing in vitro data

6a

Has the substance demonstrated corrosive properties in an EU/OECD adopted in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but
are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met

NO

 

6b

Has the substance demonstrated irritant or non-irritant properties in an EU/OECD adopted
in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but
are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are
met.

NO

(at the initiation of the dossier, no test was available)

6c

Are there data from a non-validated suitable in vitro test(s), which provide sound conclusive evidence that the substance is corrosive/ irritant?

NO

 

Weight-of- Evidence analysis

7

The “elements” described above may be arranged as appropriate. Taking all available existing and
relevant data mentioned above (Elements 1-6) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and –if so –how to classify and label?

NO

 

New in vitro test for corrosivity

8

Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

NO

Based on all available data (ATD, LLNA, QSARs), the substance is not considered as a skin corrosive => a skin corrosion assay was not required

New in vitro test for irritation

9

Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

YES

 => an Episkin test for irritation was initiated.
Viability = 52,2% & IL-alpha = 26,15 pg/mL <=> Not a skin irritant (borderline)

=> an Epiderm test for irritation was performed.

The conclusion of this Epiderm test is negative, not a skin irritant. Viability = 94.3%.

New in vivo test for serious eye damage/eye irritation as a last resort (Annex VIII to the REACH Regulation)

10

Does the substance demonstrate corrosive or irritant properties in an EU/OECD adopted in vivo test?

NO

In vivo testing should not be conducted in this case since the substance falls under the scope of the specific in vitro tests performed, and there are no substance-specific limitations on use of those tests.

A new in vitro test was performed on the substance (Envigo, 2018a). This Episkin study was performed according to the OECD Guideline 439 and in compliance with GLP. The substance with a mean tissue viability of 52.2 ± 5.6 % and a mean IL-alpha concentration = 26.15 , was predicted as not irritant to the skin (borderline).

To confirm this result, another in vitro test was performed (Envigo, 2018b). This Epiderm study was performed according to the OECD Guideline 439 and in compliance with GLP. The substance, with a mean tissue viability of 94.3%, was confirmed to be non irritant to the skin.

Eye irritation:

Since no key study was identified on the substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the eye corrosion/irritation potential of the substance:

 

Element

Information

Conclusion

Comments

Conclusion of the information strategy on skin corrosion/irritation

0

Is the substance classified as a skin corrosive?

NO

 

Existing data on physico
- chemical properties

1a

Is the substance spontaneously flammable in contact with air (pyrophoric) or water at room temperature?

NO

 

1b

Is the substance an organic hydroperoxide or an organic peroxide?

NO

 

1c

Is the pH of the substance ≤ 2.0 or ≥ 11.5?

NO

 

1d

Are there other physical or chemical properties that indicate that the substance causes serious eye damage or eye irritation?

NO

 

Existing human data

2

Are there adequate existing human data which provide evidence that the substance has the potential to cause serious eye damage or eye irritation?

NO

 

Existing animal data from corrosion/irritation studies

3

Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?

NO

 

Existing/new (Q)SAR data and read-across

4

Are there structurally related substances (suitable “read-across” or grouping), which are classified as causing serious eye damage/eye irritation, or indicating that the substance is non-irritant, or do valid (Q)SAR methods indicate serious eye damage/eye irritation or non-irritation of the substance?

NO

"Not irritating" using Toxtree (v2.6.6).
OASIS TIMES (v2.27.17) did not indicate any eye irritation alert but the prediction was not supported by sufficient applicability domain.

Existing in vitro data

5a

Has the substance demonstrated serious eye damage, eye irritation or non-irritating properties in an EU/OECD adopted in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

NO

 

5b

Are there acceptable data from (a) non-validated suitable in vitro test(s), which provide sound evidence that the substance causes serious eye damage/eye irritation?

NO

(at the initiation of the dossier, no test was available)

Weight-of- Evidence analysis

6

The “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 0 – 5) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and – if so – how to classify and label?

NO

 

New in vitro tests for serious eye damage/eye irritation (Annex VII to the REACH Regulation)

7a

Does the substance demonstrate serious eye damage, eye irritation or non-irritant properties in (an) EU/OECD adopted in vitro test(s) for the eye hazard charaterisation?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions of Annex XI are met.

NO

 

7b

Does the substance demonstrate serious eye damage or eye irritant properties in (a) non-validated suitable in vitro test(s) for serious eye damage/eye irritation?

NO

 => an ICE assay was initiated.
The conclusion of this test is no prediction can be made (IVIS = 12.8)
<=> another in vitro study was performed (EpiOcular - OECD 492). Not an eye irritant (viability = 100.7%)

New in vivo test for serious eye damage/eye irritation as a last resort (Annex VIII to the REACH Regulation)

8b

Does the substance demonstrate serious eye damage or eye irritation in an OECD adopted in vivo test?

NO

In vivo testing should not be conducted at Annex VII level

A new in vitro study was performed on the substance (Envigo, 2018a, rel.1). An ICE study was performed according to the OECD Guideline 438 and in compliance with GLP.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 2.0 (180-240 min.), corresponding to the ICE class III;

- mean score of fluorescein retention: 0.7 corresponding to the ICE class II;

- maximal mean corneal swelling: 13.08% (75 min.) corresponding to the ICE class III.

The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Under the test conditions, no prediction can be made for the test substance.

A second in vitro test was therefore performed on the substance (Envigo, 2018b, rel.1). An EpiOcular study was performed according to the OECD 492 and in compliance with GLP. With a viability of 100.7%, the test item does not possess any eye irritation potential. The quality of the tissues and the validity of the test system were confirmed by the results if the negative and positive controls.

Based on available data, the substance is considered to be not irritant to eyes.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

Skin irritation:

Based on the available information, no additional self-classification is proposed regarding skin irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

Eye irritation:

Based on the available information, no additional self-classification is proposed regarding eye irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

 

Respiratory irritation:

No data was available.