Registration Dossier

Administrative data

Description of key information

Skin Corrosion: Not corrosive; OECD 431; Spohr, C. (2018)

Skin Irritation: Not irritant; OECD 439: Spohr, C. (2018)

Eye Irritation/Corrosion: Not irritant; OECD 437; Spohr, C. (2018)

Eye Irritation: Not irritant; OECD 492; Spohr, C. (2018)

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:
01 - 24 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 Jul 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Adopting the 27th time to technical progress the Dangerous Substances Directive 67/548/EEC, Annex V, part B40 and EC regulation No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation No 1907/2006 of the European Parliament and of the Council on REACH, 1st ATP, section B40.
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek test protocol “In vitro EpiDerm Skin Corrosion Test (EPI-200-SCT)”
Version / remarks:
07 Nov 2014
Deviations:
yes
Remarks:
The formazan salt extraction period was extended up to 72 hours. According to an expert statement (from MatTek Corporation) this procedure does not have an impact on the outcome of the study.
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm tissues supplied by MatTek Corporation, Slovakia.
Justification for test system used:
Guideline specific test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 288647
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 21 Aug 2018
- Date of initiation of testing: 21 Aug 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature ( 3 min exposure group) and 37 ± 1.5 ºC (60 min exposure group)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h incubation with MTT solution followd by 19 h extraction.
- Spectrophotometer: Microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570)
- Wavelength: 570 nm
- Filter: No
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Acceptable (2.097 ± 0.214). Acceptance criteria = 1.0 - 3.0.
- Barrier function: Acceptable (6.33 h). Acceptance criteria 4.77 - 8.72 h.
- Morphology: Acceptable.
- Contamination: Acceptable (sterile).
- Reproducibility: COV of mean replicates = < 30 %.

NUMBER OF REPLICATE TISSUES: 1

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : N/A
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : N/A
- Method of calculation used: N/A

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- 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:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Unchanged - applied as supplied

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
Incubated in MTT medium for 3 hours and followed by an 19 h extraction in isopropanol.
Number of replicates:
2 for each timepoint
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
97.62
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure
Value:
100.04
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes inc. certificate.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time.
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour, is <15 % compared to the negative control.
- Acceptance criteria met for variability between replicate measurements: Coefficient of Variation (CV) in the range 20 – 100 % viability between tissue replicates is ≤ 30 %.
- Range of historical values if different from the ones specified in the test guideline:
Positive control: 3 min = 4.6 - 39.83 % viability, 60 min = 1.07 - 14.77 % viability (n=33).
Negative control: 3 min = 1.25-1.93 OD range, 60 min = 1.23-2.00 OD range (n=33)

Table.2       Viabilities for the negative control, positive control and test item

 

Treatment group

Tissue no.

Exposure interval (mins)

OD 570 nm

Mean OD of 3 wells (blank corrected)

Mean OD of 2 tissues (blank corrected)

SD (well 1-3)

Mean relative viability (%)

CV (%)

Well 1

Well 2

Well 3

Blank

-

3

0.038

0.038

0.038

 

Negative control

1

2.034

1.980

2.078

1.992

1.970

0.049

100.00

1.6

2

2.035

1.967

1.954

1.947

0.043

Positive control

1

0.300

0.293

0.290

0.256

0.211

0.005

10.70

30.6

2

0.205

0.202

0.203

0.165

0.002

Test item

1

2.073

1.994

2.013

1.989

1.923

0.041

97.62

4.8

2

1.867

1.911

1.908

1.857

0.024

Blank

-

60

0.038

0.038

0.038

 

Negative control

1

2.209

2.157

2.162

2.138

2.016

0.028

100.00

8.5

2

1.959

1.928

1.912

1.895

0.024

Positive control

1

0.125

0.120

0.115

0.082

0.069

0.005

3.41

27.1

2

0.095

0.094

0.093

0.056

0.001

Test item

1

2.088

2.051

2.059

2.028

2.017

0.020

100.04

0.8

2

2.037

2.063

2.033

2.006

0.016

 

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study the test substance is not considered to be corrosive.
Executive summary:

OECD 431 (2018) - The skin corrosivity potential of the test item was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

The test item passed the MTT- and the colour interference pre-tests and all acceptability criteria were met. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (10.70%) and for the 1 hour exposure period (3.41%) thus confirming the validity of the test system and the specific batch of tissue models.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 97.62 and 100.04 %, respectively.

 

Under the conditions of this study the test substance is not considered to be corrosive to the skin according to the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixture.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August - 07 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No. 640/2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiSkin kits purchased from SkinEthic Laboratories (Lyon, France)
Justification for test system used:
Guideline specified test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin
- Tissue batch number(s): 18-EKIN-036
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 04 Sep 2018
- Date of initiation of testing: 04 Sep 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 ºC
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Volume of washing not reported.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h followed by a 2.5 h extraction in IPA.
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Softmax Pro Enterprise)
- Wavelength: 570 nm
- Filter: 570 nm
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology scoring: 22.8 ± 0.5 (cv % = 2.3 %). Well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum. (Specification = ≥19.5)
- IC 50 determination: 2.0 mg/mL (Specification 1.5 mg/mL ≤ IC50 ≤ 3.0 mg/mL)
- Biological safety: Absence of HIV1/2 and hepatitis C antibodies and hepatitis B antigen HB's confirmed. Absence of bacteria, fungus and mycoplasm confirmed.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : N/A
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : N/A
- Method of calculation used: N/A

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure is less than 50 %, realtive to the negative control.
- The test substance is considered to be non-irritant to skin if the viability after exposure is greater than or equal to 50 %, relative to the negative control.
- 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:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): N/A - applied undiluted, as supplied.

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5 %
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
Post treatment incubation: 42 h
MTT assay: 3 h incubation followed by 2.5 h extraction.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean tissue viability (%)
Run / experiment:
15 min exposure
Value:
116.7
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:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The OD values for the negative controls were between 0.6 and 1.5 (0.643 to 0.692).
- Acceptance criteria met for positive control: Viability of tissues was ≤ 40 % (13 %).
- Acceptance criteria met for variability between replicate measurements: Yes, ≤18 % (ca. 4 % for both negative and positive control groups).
- Range of historical values if different from the ones specified in the test guideline: The results for the positive and negative controls are within the historical data (means, rel. standard deviation, and ranges) of Envigo CRS GmbH.

Table 2       OD570 values and viabilities for the negative control, positive control and test item

 

Treatment group

Tissue no.

OD570 nm

Mean OD of wells (blank corrected)

Mean OD of 3 tissues (blank corrected)

Relative viability

(%)

RSD (%)

Mean relative viability (%)

Well 1

Well 2

Mean

Blank

-

0.038

0.038

0.038

-

-

-

-

-

Negative control

1

0.705

0.689

0.697

0.659

0.664

99.1

3.8

100.0

2

0.745

0.714

0.730

0.692

104.1

3

0.689

0.672

0.681

0.643

96.7

Negative control

1

0.139

0.134

0.136

0.098

0.086

14.8

4.7

13.0

2

0.151

0.144

0.148

0.110

16.5

3

0.090

0.088

0.089

0.051

7.7

Negative control

1

0.826

0.774

0.800

0.762

0.775

114.7

3.4

116.7

2

0.862

0.817

0.840

0.802

120.7

3

0.820

0.781

0.800

0.762

114.8

 

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study the test substance is not considered to be irritant to the skin and is not classifed accoring to UN GHS and EU CLP Regulation.
Executive summary:

OECD 439 (2018) - The skin irritation potential of the test item was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

 

Triplicate tissues were exposed to the test item for 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

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

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

Mean viability of tissues exposed to the test substance after 15 minutes were 116.7 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the results were met.

 

Under the conditions of this study the test substance is not considered to be irritant to the skin and is not classified in accordance with UN GHS and EU CLP Regulation (EC) No. 1272/2008.

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:
10 August - 10 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
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
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) No 1152/2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A - test item applied undiluted.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A - test item applied undiluted.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A - test item applied as supplied.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A

OTHER SPECIFICS:
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): Cattle were ≥ 9 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: Same day testing.
- indication of any existing defects or lesions in ocular tissue samples: Only cornea free of defects were used in the test.
- Indication of any antibiotics used: HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) during storage and shipping of isolated eyes.
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): 0.75 mL
- Concentration (if solution): N/A - test item applied undiluted.

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A - vehicle not used.
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Duration of treatment / exposure:
10 mins ± 30 seconds
Observation period (in vivo):
N/A
Duration of post- treatment incubation (in vitro):
120 mins - for second opacity measurement (defined as t130).
A further 90 mins - for permeability determination.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O- ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0). At the end of the incubation period, the basal opacity was determined (t0).

QUALITY CHECK OF THE ISOLATED CORNEAS : Only corneae with a value of the basal opacity < 7 were used.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : Yes (n=3), Saline (0.9% NaCl in deionised water)

SOLVENT CONTROL USED (if applicable) : N/A

POSITIVE CONTROL USED : Yes (n=3), 2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME : The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL on the surface of the corneae via open chamber method, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes (± 30 seconds).

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: For opacity measurements - After the test item or control items, respectively, were rinsed off from the application side with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium is free of test item the corneas are given a final rinse with cMEM without phenol red. Fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130).

For permeability determination - Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least 3 times.
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: To determine light transmission through tthe cornea, an opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was used.
- Corneal permeability: The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490).
- Others (e.g, pertinent visual observations, histopathology): (please specify) N/A

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean IVIS (n=3)
Value:
0.04
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, values of 65 studies with liquid test items sharing 37 sets of controls, performed between January 2016 and July 2018 reported.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Tthe negative control responses resulted in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: The positive control indicated an IVIS that falls within two standard deviations of the current historical mean (updated every three months).
- Range of historical values if different from the ones specified in the test guideline: N/A

Table 1       IVIS scores

 

Test Group

Rep.

Opacity value

(t130– t0)

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Negative control

1

0.00

0.056

0.84

0.97

2

0.00

0.062

0.93

3

0.00

0.075

1.13

Mean

0.00

0.064

-

Positive control

1

89.00

0.697

99.45

102.35

2

88.00

0.611

97.16

3

101.00

0630

110.45

Test item

1

0.00

-0.002

-0.04

0.04

2

0.00

-0.002

-0.04

3

0.00

0.013

0.19

*corrected values

Interpretation of results:
GHS criteria not met
Conclusions:
According to the current study and under the experimental conditions reported, the test item does not meet the GHS classification criteria.
Executive summary:

OECD 437 (2018) - The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using the test item in accordance with 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" (2013).

Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared. Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 30 min incubation in the vertical position with sodium fluorescein. Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490).

The quality criteria required for acceptance of the results were met.  The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/UN GHS (Cat 1)).

The mean corrected opacity reading and permeability readings for the test item were 0.00 and 0.003, resulting in an In Vitro Irritation Score (IVIS) of 0.04.

According to the current study and under the conditions of the experimental conditions reported, the test item does not meet the criteria categorisation in accordance with UN GHS and EU CLP Regulation (EC) No. 1272/2008

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 - 30 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guideliens and in accordance with GLP. All guidelines validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
October 2017
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcularTM Model
Version / remarks:
June 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals or tissues and environmental conditions:
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 (MILLICELL, 10 mm ).

EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (28 August 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 of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (about 20.5 hours).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): N/A - applied undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
Post treatment: 120 minutes
MTT assay: 180 minutes incubation followed by 120 minute extraction period.
Number of animals or in vitro replicates:
2
Details on study design:
Test item exposure

After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++Mg++free- DPBS. Since the Ca++Mg++free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) for 30 minutes.
After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 μL topically on the EpiOcularTM tissues. The tissues were incubated at standard culture conditions for 30 minutes.
At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beaker of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled microtiter plate containing 1 mL of warm Assay Medium. The tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

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 parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were stored overnight at 2- 8 °C in the dark and then shaken for 2 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.
Irritation parameter:
other: Spectrophotometric mean relative absorbance of MTT solution at 570 nm
Run / experiment:
Mean relative tissue viability (%)
Value:
104.12
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported

ACCEPTANCE OF RESULTS:
- The negative control OD is > 0.8 and < 2.5 (2.304 - 2.528 (mean values = 2.310 - 2.494)).
- The mean relative viability of the positive control is below 50% of the negative control viability (46.58 %).
- The difference of viability between the two relating tissues of a single item is < 20% (values between 6.05 and 7.78 %) in the same run (for positive and negative control tissues and tissues of single test items).

Table 1       OD570 measurement and tissue viability values for negative control, positive control and test item

 

Test group

Tissue no.

Well 1

(OD570)

Well 2

(OD570)

Mean (OD570) (Well 1 & 2)

Mean (OD570) blank corrected (Well 1 & 2)

Mean (OD570) of Rep 1 & 2

Tissue viability (%)

Relative viability of Rep 1 and Rep 2

Difference of viability between Rep 1 and Rep 2 (%)

Blank

-

0.036

0.035

0.036

-

-

-

-

-

Negative control

1

2.460

2.528

2.494

2.458

2.366

100.0

103.9

7.78

2

2.316

2.304

2.310

2.274

96.1

Positive control

1

1.027

1.106

1.066

1.031

1.102

46.58

43.6

6.05

2

1.195

1.224

1.209

1.174

49.6

Test item

1

2.299

2.524

2.411

2.375

2.464

104.12

100.4

7.45

2

2.587

2.589

2.588

2.552

107.8

Interpretation of results:
GHS criteria not met
Conclusions:
In this study and under the conditions of the experiment, the test item does not possess eye irritating potential.
Executive summary:

OECD 492 (2018) - This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol and did not prove to be an MTT reducer.

 

Tissues of the human cornea model EpiOcular™ were treated with the test item, the positive and the negative control for 30 minutes each in duplicate. 50 µL of the test item and of the controls, respectively, were applied to each tissue, spread to match the tissue size.

 

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 46.58 %, thus the validity of the test system is ensured. Irritating effects were not observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability were 104.12 % compared with the value of the negative control (threshold for irritancy: ≤ 60%).

 

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess an eye irritating potential and does not meet classification criteria according to the CLP Regulation (EC) No. 1272/2008 and UN GHS.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion/ irritation;

OECD 431 (2018) - The skin corrosivity potential of the test item was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

 

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 97.62 and 100.04 %, respectively. The quality criteria required for acceptance of the results was met.

 

Under the conditions of this study the test substance is not considered to be corrosive to the skin.

OECD 439 (2018) - The skin irritation potential of the test item was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

 

Triplicate tissues were exposed to the test item for 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

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

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

Mean viability of tissues exposed to the test substance after 15 minutes were 116.7 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the results were met.

 

Under the conditions of this study the test substance is not considered to be irritant to the skin and is not classified in accordance with UN GHS and EU CLP Regulation (EC) No. 1272/2008.

Eye corrosion/irrittion

OECD 492 (2018) - This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol and did not prove to be an MTT reducer.

 

Tissues of the human cornea model EpiOcular™ were treated with the test item, the positive and the negative control for 30 minutes each in duplicate. 50 µL of the test item and of the controls, respectively, were applied to each tissue, spread to match the tissue size.

 

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 46.58 %, thus the validity of the test system is ensured. Irritating effects were not observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability were 104.12 % compared with the value of the negative control (threshold for irritancy: ≤ 60%).

OECD 437 (2018) - The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using the test item in accordance with 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" (2013).

Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared. Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 30 min incubation in the vertical position with sodium fluorescein. Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490).

The quality criteria required for acceptance of the results were met.  The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/UN GHS (Cat 1)).

The mean corrected opacity reading and permeability readings for the test item were 0.00 and 0.003, resulting in an In Vitro Irritation Score (IVIS) of 0.04.

According to the current study and under the conditions of the experimental conditions reported, the test item does not meet the criteria categorisation in accordance with UN GHS and EU CLP Regulation (EC) No. 1272/2008

Justification for classification or non-classification

Skin corrosion - Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 97.62 and 100.04 %, respectively. The quality criteria required for acceptance of the results was met. Under the conditions of this study the test substance isn’t considered to be corrosive to the skin.

Skin irritation - Mean viability of tissues exposed to the test substance after 30 minutes were 104.12 %. The quality criteria required for acceptance of the results was met. Under the conditions of this study the test substance was not considered to be irritant to the skin and does not meet the classification criteria according to UN GHS and EU CLP regulation.

Eye Irritation/corrosion - The mean corrected opacity reading and permeability readings for the test item were 0.00 and 0.003, resulting in an In Vitro Irritation Score (IVIS) of 0.04 As a result the test substance was not considered to be irritant to the eye and does not meet the classification criteria according to UN GHS and EU CLP regulation

Eye Irritation - In vitro eye irritation was investigated further under OECD 492. The mean relative absorption value of the tissues corresponding to the cornea viability was 104.12 % compared with the value of the negative control (threshold for irritancy: ≤ 60%), concluding that the test substance was not considered to be irritant to the eye and does not meet the classification criteria according to UN GHS and EU CLP regulation.