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Diss Factsheets

Administrative data

Description of key information

The test substance showed to be irritating to the skin and eye.

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:
weight of evidence
Study period:
30 Nov 2015 to 25 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 28 July 2015
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
Official Journal of the European Union, No. L 142
30 May 2008
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Purity test date: 100 % UVCB
- Homogeity: The test substance was homogeneous by visual inspection.
- Appearance: Solid / beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Test system:
human skin model
Remarks:
EpiDerm(TM) model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: cultured
Vehicle:
unchanged (no vehicle)
Details on test system:
MATERIALS AND TECHNICAL EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220
- Incubation conditions: 37°C ± 1°C, 5% ± 1%CO2, 90% ± 5%humidity
- Spectrophotometer: SunriseTM Absorbance Reader For the determination of the optical density of colored extracts.Measurement using a filter wavelength 570 nm without reference filter
- EpiDerm™ 200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia; containing: 24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® ø 1 cm
- Assay medium: EPI-100-NMM assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

TEST SYSTEM
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
- Tissue model: EPI-200
- Tissue Lot Number: 23305
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

DIRECT MTT REDUCTION
- The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
- To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as described below.
- The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently.
- If the color of the MTT solution or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
- In case that direct MTT reduction occurred, two freeze-killed control tissue (KC) per exposure time were treated with, the test article and the negative control.

BASIC PROCEDURE
Several test substances were tested in parallel within the present test using the same control tissues (NC and PC).
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6- well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used.
25 μL de-ionized water was applied first. Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water. Control tissues were concurrently treated with 50 μL of de-ionized water (NC) or with 50 μL of 8 N potassium hydroxide (PC).
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.
Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria was not met, repetition of the test was considered.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines. Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Acceptance criteria for the negative control (NC): The absolute OD570 of the negative control tissues in the MTTtest is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria for the positive control (PC): Potassium hydroxide as 8.0 normal ready-made solution is used as positive reference. A tissue viability of ≥ 30%
is acceptable for the 3-minute exposure. Mean viability of the tissues exposed for 1 hour should be <15%.
- Acceptance criteria for the variability of the tissues: For every treatment two tissues are treated in parallel. In the range of 20% and 100% viability, variability between the tissues is considered to be acceptable if the coefficient of variation (CV) of %-viability is ≤ 30%.

EVALUATION OF RESULTS
- Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
- A single test composed of at least two tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as nonconcordant replicate measurements and/or mean percent tissue viability equal to ±5% of the cut-off values cited above, a second test should be considered, as well as a third one in case of discordant results between the first two tests.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
25 μL de-ionized water was applied first (vehicle). Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.

CONTROLS
Control tissues were concurrently treated with 50 μL of de-ionized water (NC) or with 50 μL of 8 N potassium hydroxide (PC).
Duration of treatment / exposure:
3 min and 1 hour
Duration of post-treatment incubation (if applicable):
42 hour post-incubation period
Number of replicates:
2 replicates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Corrosion test: 3 min exposure
Value:
97.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Corrosion test: 1 hour exposure
Value:
73.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
In combination with the irritation results (OECD 439)
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
30 Nov 2015 to 25 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
6 July 2012
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Purity test date: 100 % UVCB
- Homogeity: The test substance was homogeneous by visual inspection.
- Appearance: Solid / beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Test system:
human skin model
Remarks:
EpiDerm(TM) model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: cultured
Vehicle:
unchanged (no vehicle)
Details on test system:
MATERIALS AND TECHNICAL EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220
- Incubation conditions: 37°C ± 1°C, 5% ± 1%CO2, 90% ± 5%humidity
- Spectrophotometer: SunriseTM Absorbance Reader For the determination of the optical density of colored extracts.Measurement using a filter wavelength 570 nm without reference filter
- EpiDerm™ 200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia; containing: 24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® ø 1 cm
- Assay medium: EPI-100-NMM assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

TEST SYSTEM
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
- Tissue model: EPI-200
- Tissue Lot Number: 23305
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

DIRECT MTT REDUCTION
- The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
- To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as described below.
- The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently.
- If the color of the MTT solution or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
- In case that direct MTT reduction occurred, three freeze-killed control tissue (KC) were treated with, the test article and the negative control

BASIC PROCEDURE
Several test substances were tested in parallel within the present test using the
same control tissues (NC and PC).
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively.
25 μL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
Control tissues were concurrently treated with 30 μL of sterile PBS (NC) or with 30 μL of 5% SDS (PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6- well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria was not met, repetition of the test was considered.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines. Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Acceptance criteria for the negative control (NC): The absolute OD570 of the negative control tissues in the MTTtest is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≥ 20% is acceptable.
- Acceptance criteria for the variability of the tissues: For every treatment three tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 18%.

EVALUATION OF RESULTS
- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
- A single test composed of at least three tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as nonconcordant replicate measurements and/or mean percent tissue viability equal to ±5% of the cut-off value, a second test should be considered, as well as a third one in case of discordant results between the first two tests.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
25 μL sterile PBS was applied first (vehicle). Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.

CONTROLS
Control tissues were concurrently treated with 30 μL of sterile PBS (NC) or with 30 μL of 5% SDS (PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hour post-incubation period
Number of replicates:
3 replicates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Irritation test: 1 hour exposure
Value:
18.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
In combination with the skin corrosion results (OECD 431)
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
02 Nov 2016 to 05 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
October 02, 2012
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
30 May 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
August 1998
Qualifier:
according to guideline
Guideline:
other: Japan MAFF Testing Guideline of 12 Nousan No. 8147
Version / remarks:
November 24, 2000
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Test item No.: 15/0531-1
- Purity test date: 100% UVCB
- Homogeity: The test substance was homogeneous by visual inspection.
- Appearance: Solid / beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hsdlf:NZW – (SPF)
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Envigo CRS (Switzerland) Limited
- Sex: female
- Age at study initiation: Approx. 4 months
- Weight at study initiation: 2.96 – 3.54 kg
- Housing: Animals were individually housed in stainless steel wire mesh cages with grating with shallow cage body; floor area: 4225 cm2.
- Enrichment: Wooden gnawing blocks (Type KNH E-041); Abedd ® Lab. and Vet. Service GmbH Vienna, Austria
- Diet: STANRAB (P) SQC; SDS Special Diets Services, 67122 Altrip, Germany, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 30-70 %
- Air changes: Approx. 10
- Photoperiod: 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 mL bulk volume (about 28 mg of the comminuted test item).
Duration of treatment / exposure:
The test item was applied in a single dose to the conjunctival sac of the right eyelid.
Observation period (in vivo):
Approx. 1, 24, 48 and 72 h after application and then at weekly intervals maximally up to day 14.
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: the treated eye of the animal(s) was rinsed with 3 to 6 mL of lukewarm tap water for 1 to 2 minutes using a syringe with a blunt probe.
- Time after start exposure period: about (but not less than) 1 hour after application of the test item

PROCEDURE
- Body weight determination: Just before application of the test item and after the last reading.
- Local anesthesia: One drop of local anesthetic eye drops in each eye.
- Analgesia: 1 hour before and 8 hours after application with buprenorphine (0.01 mg/kg s.c.) and thereafter in 12 hour intervals until local and/or systemic findings had nearly disappeared. Meloxicam (0.5 mg/kg s.c.) 8 hours after application and thereafter in 24 hours intervals until local and/or systemic findings had nearly disappeared.
- Mortality: A check for any dead or moribund animals was made at least once each day.

SCORING SYSTEM:
The evaluation of eye irritation was performed according to the quoted guidelines. In addition to specific observations recommended by the regulatory authorities, evaluations were made of discharge from the eye and the area of cornea affected by the lesions.

TOOL USED TO ASSESS SCORE:
Reading reactions on cornea or iris were observed using a slit lamp. Additional findings like corneal lesions detected with the aid of fluorescein.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
iris score
Basis:
animal: #1 and #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal: #1 and #2
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
Additional findings like corneal lesions detected with the aid of fluorescein (grade 1 – 2) and injected scleral vessels in a circumscribed or circular area were noted in the animals within 7 days after application.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
02 Dec 2015 to 25 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
28 July 2015
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Test-substance No.: 15/0531-1
- Purity test date: 100 % UVCB
- Homogeity: The test substance was homogeneous by visual inspection.
- Appearance: Solid / beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Species:
human
Details on test animals or tissues and environmental conditions:
TISSUE MODEL
- Tissue model: OCL-200 (EpiOcularTM model)
- Tissue Lot Number: 21588 (test run 1) and 21593 (test run 2)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

MATERIALS AND TECHNICAL EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION) CO2 incubator: Heraeus BBD 6220
- Incubation conditions: 37°C ± 1°C, 5% ± 1% CO2, 90% ± 5% humidity
- Spectrophotometer: SunriseTM Absorbance Reader for the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter
- EpiOcular™ OCL-200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia. Containing: 24 OCL-200 tissues (reconstructed cornea): surface 0.6 cm² cultured in Millicells® (1 cm diameter).
- Assay medium: OCL-200-ASY assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
- Pre-treatment / wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
- Using a sharp spoon, a bulk volume of ca. 50 μL of the test material was applied covering the whole tissue surface.
- Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC).
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
TISSUE PREPARATIONS
- On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
- After the pre-incubation, the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
- After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

DIRECT MTT REDUCTION
- To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
- The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
- In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.

BASIC PROCEDURE
- Several test substances were tested in parallel within the present tests using the same control tissues (NC and PC) within the respective test. Two tissues were treated with each, the test substance, the PC and the NC.
- There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical state of the test substance the protocol for solids was applied.

PRE-INCUBATION OF THE TISSUES
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

PRETREATMENT OF THE TISSUES
After the pre-incubation, the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

POST TREATMENT
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.

MTT INCUBATION
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation ofthe tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

METHODS FOR MEASURED ENDPOINTS
- Principle: The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material was an irritant.
- Calculation of individual and mean optical densities: The corrected measured OD570 value for each individual tissue was calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way was calculated.
- Tissue viability: The quantification of tissue viability is presented as the ratio of the mean OD570 divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
- Barrier funciton and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 (min) value following exposure to 100 μL of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200) batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guideline. Lower acceptance limit: ET50 = 12.2 min; Upper acceptance limit: ET50 = 37.5 min
- Acceptance criteria for the NC: The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is > 0.8. The mean OD570 of the NC should not exceed 2.5.
- Acceptance citeria for the PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Acceptance criteria for tissue variability: Two tissues were treated under the same conditions. A variability between the the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.

EVALUATION OF RESULTS
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60%.

A single test composed of at least two tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as nonconcordant replicate measurements and/or mean percent tissue viability equal to ±5% of the cut-off value, a second test should be considered, as well as a third one in case of discordant results between the first two tests. See 'Any other information on materials and methods incl. tables'

Irritation parameter:
other: % viability [% of NC]
Run / experiment:
1
Value:
1.6
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
other: % viability [% of NC]
Run / experiment:
2
Value:
1.6
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS
Detachment and/or damage of the tissues, leading to very low viability values, were noted during the washing procedure of the 1st and 2nd test run. Thus, the 2nd test run verified the results of the 1st test run and the destruction of the tissues is attributed to the irritant effect of the test substance.

ACCEPTANCE OF RESULTS
- Acceptance criteria for negative control are met
- Acceptance criteria met for positive control: The viability value of the PC (1st test run) of the present study lies minimally out of the range of the data given above. However, as all other quality criteria of the test were met, this deviation is not considered to have any influence on the validity of the data.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
02 Dec 2015 to 25 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July 2013
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
8 December 2010
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Test-substance No.: 15/0531-1
- Purity test date: 100 % UVCB
- Homogeity: The test substance was homogeneous by visual inspection.
- Appearance: Solid / beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Species:
cattle
Strain:
other: bovine
Details on test animals or tissues and environmental conditions:
TISSUE MODEL
- Isolated bovine cornea: The test system is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
- Supplier: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany

MATERIALS AND TECHNICAL EQUIPMENT
- Corneal holder: Supplier: BASF SE, Germany
- Incubator: Temperature 32 ± 1 °C
- Opacitometer: Kit BASF-OP3.0, BASF SE, Germany
- Spectrophotometer: SunriseTM Absorbance Reader Measurement using wavelength of 490 nm

REAGENTS
- Hanks' Balanced Salt Solution with Ca++ and Mg++ (HBSS) (Biochrom, Germany) containing Fetal Bovine Serum (FBS) and/or Penicillin/Streptomycin (P/S)
- Eagle’s MEM without phenol red (Biochrom, Germany) containing FBS and P/S
- Eagle´s MEM with phenol red (Biochrom, Germany)
- Sodium fluorescein diluted in DPBS
Vehicle:
water
Remarks:
de-ionized
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Concentration: 10% (w/v) suspension in de-ionized water
- Amount: 750 µL
Duration of treatment / exposure:
Approximately 10 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
- After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 544 opacity units1 were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups. Each corneal holder was uniquely identified with a number on the chambers.

APPLICATION
- Before application, the medium in the anterior chamber was removed using a syringe.
- 750 µL of the 10% (w/v) test-substance preparantion was applied into the anterior chamber using a pipette
- For the control tissues 750 μL of de-ionized water (negative control, NC) or 750 μL of 100% ethanol / 100% dimethylformamide (positive control, PC1 / PC2), were applied into the anterior chamber using a pipette.
- The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
- Post-exposure incubation: The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Permeability: For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min. in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.
- Histopathology: After determination of opacity and permeability, the corneas were fixed in 4% formaldeyhde for at least 24 h and transferred to the laboratory of General Pathology for further histotechnical processing and examination by light microscopy. Histopathological findings were summarized in a histopathological score of irritation (HSI).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

ACCEPTANCE CRITERIA
- A study is considered acceptable if the PC gives an IVIS that falls within two standard deviations of the current historic mean.
- The NC responses should result in opacity and permeability values that are not higher than the established upper limits.
- Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. In cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs. In this context, a result in the first testing run is considered borderline if the predictions from the 3 corneas were non-concordant, such that:
* 2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
* 1 of the 3 corneas gave a discordant prediction from the mean of all 3 corneas, AND thediscordant result was >10 IVIS units from the cut-off threshold of 55.

DECISION CRITERIA
See 'Any other information on materials and methods incl. tables'
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean IVIS score
Value:
2.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Mean corrected opacity change
Value:
1.1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
Mean corrected OD490
Value:
0.085
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
histopathological observations
Run / experiment:
Mean histological evaluation score
Value:
2
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control are met
- Acceptance criteria met for positive control: The permeability value of the positive control PC1 (100% ethanol) is out of the historical range. However, this is attributed to the limited number of historic control data available at present. Due to the unambiguous result of the study this deviation is not considered to have any influence on the validity of the data.
Interpretation of results:
study cannot be used for classification
Remarks:
The BCOP test is unsuitable for classification in case of a negative outcome
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion: in vitro

Two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

Skin corrosion

Skin corrosive potential was assessed in the Skin Corrosion Test (SIT) according to OECD TG 431 under GLP conditions (BASF, 2016). A total of two tissues of the reconstructed three dimensional human epidermis model (EpidermTM) were exposed to a single topical application of 25 µL bulk volume (ca. 11 mg) of the undiluted test substance for 3 minutes and 1 hour, each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 97.9%, and it was 73.5% after an exposure period of 1 hour.

Skin irritation

Skin irritation potential was assessed in the Skin Irritation Test (SIT) according to OECD TG 439 under GLP conditions (BASF, 2016). A total of three tissues of the reconstructed three dimensional human epidermis model (EpidermTM) were exposed to a single topical application of 25 µL bulk volume (ca. 11 mg) of the undiluted test substance for 1 hour followed by a 42 -hours post icubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 18.4%.

Based on the results of both studies, it was concluded that the test substance shows a skin irritation potential.

Eye irritation: in vitro

Two in vitro (BCOP and EpiOcular assay) studies were of the in vitro eye irritation and corrosion test strategy.

BCOP

Eye irritation potential was assessed in the Bovine Corneal Opacity and Permeability Test (BCOP Test) according to OECD TG 437 under GLP conditions (BASF, 2016). A total of three corneas were exposed to a single topical application of 750 µL of a 10% test-substance preparation (surfactant) for 10 minutes followed by an post-incubation period of 2 hours. De-ionized water was used as a negative control. 100% ethanol and 100% dimethylformamide were used as positive controls. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The test substances showed a mean opacity value of 1.1, a mean permeability value of 0.085 and a mean in vitro irritancy score of 2.4.

Applying the criteria of the OECD 437 for the assessment of the BCOP test results, the mean IVIS score of the test substance of 2.4 would classify it as a substance that does not cause eye irritation or serious eye damage. However, the Guideline also states, that due to limited accuracy of the BCOP test to correctly identify substances that do not require classification for eye irritation or serious eye damage, this test method should not be the first choice to initiate a bottom-up approach. Based on this statement and the experience of the test facility, test substances not leading to the prediction “ocular corrosive or severe irritant” are generally examined in the EpiOcular test as well.

EpiOcular

Eye irritation potential was assessed in the EpiOcular test according to OECD TG 492 under GLP conditions (BASF, 2016). Two EpiOcular tissues per test run were exposed to a single topical application of 50 µL bulk volume (ca. 15 mg) of the undiluted test substance for 6 hours followed by an post-incubation period of 18 hours. Two test runs were performed. Tissue destruction was determined by measuring the metabolic activity of the tissue after posure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 1.6%. The EpiOcular test was positive and the test substance was assessed to be irritating.

Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria of this combination strategy, the test substance shows an eye irritation potential in the in vitro eye irritation test strategy under the conditions chosen. Due to a negative outcome in the BCOP and a positive outcome in the EpiOcular, a confirmatory in vivo study was conducted.

Eye irritation: in vivo

An in vivo test, according to OECD 405 under GLP conditions, was performed to assess the eye irritation/corrosion potential of the test substance (Bioassay, 2017). The eye of three rabbits were exposed to 0.1 mL bulk volume (ca. 28 mg) of the undiluted test substance for 1 hour, before the eyes were rinsed. The ocular reactions were assessed approximately 1, 24, 48 and 72 hours after application and at weekly intervals until day 14. Additional eye examinations were performed following the instillation of a fluorescein solution starting at 24 h after administration until day 7 (reversibility of corneal lesions) at the latest. The following clinical substance-related observations were recorded: slight corneal opacity, moderate iritis, slight to obvious conjunctival redness, slight to moderate conjunctival chemosis and slight to severe discharge. Additional findings like corneal lesions detected with the aid of fluorescein (grade 1 – 2) and injected scleral vessels in a circumscribed or circular area were noted in the animals within 7 days after application. All reactions were reversible within 14 days. Mean scores calculated for each animal over 24, 48 and 72 hours were 0.7, 1.0 and 0.3 for corneal opacity, 0.7, 0.7 and 0.0 for iris lesions, 2.0, 2.0 and 1.7 for redness of the conjunctiva and 1.3, 2.0 and 1.7 for chemosis.

Considering the described ocular reactions and the corresponding scoring, the test substance shows an eye irritation potential under the test conditions chosen.

Justification for classification or non-classification

Based on the results of the combined testing strategy the test substance has to be classified as Skin Irrit. 2, H315: Causes skin irritation, in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

Based on the results of the combined testing strategy the test substance has to be classified as Eye Irrit. 2, H319: Causes serious eye damage, in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.