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Administrative data

Description of key information

Skin irritation test: The test material showed irritative properties

Skin corrosion test: The test material showed a corrosion potential

Bovine corneal opacity and permeability test (BCOP): no prediction can be made

EpiOcular eye irritation test: irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Details on 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 ∅) and are commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (25 min), 37°C (35 min)
- Temperature of post-treatment incubation (if applicable): 37°C

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 ul

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 ul

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 tissues
Value:
3
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Test substance identification

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

CV [%]

NC

Mean OD570

1.995

2.178

2.097

2.090

 

 

Viability [% of NC]

95.5

104.2

100.3

100.0

4.4

4.4

Test substance

Mean OD570

0.057

0.061

0.068

0.062

 

 

Viability [% of NC]

2.7

2.9

3.2

3.0

0.3

8.9

PC

Mean OD570

0.052

0.051

0.049

0.051

 

 

Viability [% of NC]

2.5

2.5

2.4

2.4

0.1

2.6

NC, negative control

PC, positive control

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Details on test system:
THREE-DIMENSIONAL HUMAN EPIDERMIS MODEL
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 ∅) and are commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
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 ground 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.

DATA EVALUATION:
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%.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 ul of solid ground material

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 ul

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 ul
Duration of treatment / exposure:
3 min or 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
86.5
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure, experiment 1
Value:
21.1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
variations in results, therefore experiment was repeated
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure, experiment 2
Value:
7.4
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid

Exposure period: 3 min

Test substance identification

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Mean OD570

1.986

2.031

2.008

 

 

Viability [% of NC]

98.9

101.1

100

1.6

1.6

Test substance

Mean OD570

1.825

1.651

1.738

 

 

Viability [% of NC]

90.0

82.2

86.5

6.1

7.1

PC

Mean OD570

0.237

0.251

0.244

 

 

Viability [% of NC]

11.8

12.5

12.1

0.5

4.1


 

Exposure period: 1 hour (Experiment 1)

Test substance identification

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Mean OD570

2.195

1.981

2.088

 

 

Viability [% of NC]

105.1

94.9

100.0

7.2

7.2

Test substance

Mean OD570

0.599

0.284

0.442

 

 

Viability [% of NC]

28.7

13.6

21.1

10.7

50.5

PC

Mean OD570

0.148

0.159

0.154

 

 

Viability [% of NC]

7.1

7.6

7.4

0.4

5.1

 

Exposure period: 1 hour (Experiment 2)

Test substance identification

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Mean OD570

1.814

1.984

1.899

 

 

Viability [% of NC]

95.5

104.5

100.0

6.3

6.3

Test substance

Mean OD570

0.136

0.146

0.141

 

 

Viability [% of NC]

7.1

7.7

7.4

0.4

5.3

PC

Mean OD570

0.078

0.087

0.082

 

 

Viability [% of NC]

4.1

4.6

4.3

0.3

7.3

 NC, negative control

PC, positive control

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
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)
GLP compliance:
yes (incl. certificate)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals (e.g. age, sex, weight): age minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: 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 opacity were discarded.
Vehicle:
water
Remarks:
deionized water
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL 20% (w/v) test-substance preparation (non-surfactant) was applied using a pipette.
- Concentration (if solution): 20% (w/v)

Duration of treatment / exposure:
The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids). 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).
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
see above

QUALITY CHECK OF THE ISOLATED CORNEAS
see above

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
see above

SOLVENT CONTROL USED (if applicable)
see above

POSITIVE CONTROL USED
see above

APPLICATION DOSE AND EXPOSURE TIME
see above

TREATMENT METHOD: [closed chamber / open chamber]
see above

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times

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.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry

SCORING SYSTEM AND DECISION CRITERIA: In Vitro Irritancy Score (IVIS)
In addition to the scoring system stated in the guideline, a borderline“-evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was determined statistically using historic BASF data. This takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437.
Irritation parameter:
in vitro irritation score
Value:
47.1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Test substance identification

Cornea No.

Opacity per cornea

Permeability per cornea

IVIS

Per cornea

Per group

Mean

SD

Test substance

10

36.4

0.504

44.0

47.1

3.1

11

35.6

0.760

47.0

12

40.4

0.652

50.2

NC

1

14.9

0.000

14.9

10.5

5.3

2

4.6

0.001

4.6

3

11.8

0.004

11.9

PC

4

74.7

4.025

135.0

130.8

3.7

5

78.0

3.336

128.0

6

86.1

2.878

129.2

NC, negative control

PC, positive control

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability:
The objective of this in vitro test is to assess the eye irritation potential of the test substance by using the reconstructed human ocular tissue model EpiOcularTM. The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three-dimensional, human cornea model EpiOcularTM. After application of the test material to the surface of the EpiOcularTM tissue, the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is spectrophotometrically determined. The optical density of the extracts of the tissues treated with the test substance is compared to values from negative control tissues and expressed as relative tissue viability.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 23724 (Certificate of Analysis see appendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 ul
Duration of treatment / exposure:
After application, the tissues were placed into the incubator until the total exposure time of
6 hours was completed.
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used:
1. 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 waterphase,
turned blue / purple, the test substance was presumed to directly reduce MTT.
2. 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.
3. Pre-treatment 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.
4. Application of the test substance
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).
After application, the tissues were placed into the incubator until the total exposure time of
6 hours was completed.
5. Removal of the test substance and postincubation period
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.
Subsequently, the tissues were incubated at standard culture conditions for 18 hours
(postincubation period).
6. 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 of
the 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.
- RhCE tissue construct used, including batch number: OCL-200, batch 23724
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable). 2
- Positive and negative control means and acceptance ranges based on historical data
The mean OD and viability value of the PC of the present EpiOcular Test were slightly below the historical range. 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.
Irritation parameter:
other: tissue viability
Run / experiment:
mean
Value:
2.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
other: The mean OD and viability value of the PC of the present Test were slightly below the historical range. 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.

Test substance identification

 

Tissue 1

Tissue 2

Mean

Inter-tissue variability [%]

NC

Mean OD570

1.594

1.557

1.575

 

 

Viability [% of NC]

101.2

98.8

100.0

2.3

Test substance

Mean OD570

0.035

0.035

0.035

 

 

Viability [% of NC]

2.2

2.2

2.2

0.0

PC

Mean OD570

0.169

0.172

0.170

 

 

Viability [% of NC]

10.7

10.9

10.8

0.2

NC, negative control

PC, positive control

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the data available and according to the criteria for classification and labelling laid down in Regulation (EC) 1272/2008 (CLP), the test substance can be considered corrosive. Therefore, classification with H314 ("causes severe skin burns and eye damage") is warranted.