Registration Dossier

Administrative data

Description of key information

The test substance is determined to be corrosive to skin.

The test substance causes serious eye damage.

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:
2017-01-17 to 2017-05-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
Version / remarks:
2015-06-28
Qualifier:
according to
Guideline:
other: New guidance document on an integrated approach on testing and assessment (IATA) for skin corrosion and irritation, Series on Testing and Assessment No. 203
Version / remarks:
2014-07-11
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Name (as cited in the study): Golpanol MPA
- Purity: 99.2 area%
- Batch Nr.: 85603856PO
- Batch Nr.:
- Contet: w(C5H9N) = 89.2 g/100 g
- Homogeneity: the test substance was homogeneous by visual inspection
- pH value: ca. 6
- Physical state / color: liquid / colorless, clear
- Storage conditions: room temperature
Test system:
artificial membrane barrier model
Remarks:
Corrositex assay
Vehicle:
unchanged (no vehicle)
Details on test system:
The Corrositex® assay was conducted according to the methods described in the Corrositex® Instruction Manual, InVitro International, Irvine CA, USA and Transia GmbH, 35510 Butzbach, Germany 10 April 2016.

- Test kit: Corrositex®, InVitro International, Irvine CA, USA, containing: reagents required for qualification and categorization screen,
biobarrier matrix powder and diluent, membrane discs and vials containing the Chemical Detection System.
- Fume hood: The assay was run in a fume hood
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- amount applied: single topical application of 500µL test substance

VEHICLE
- test substance was applied undiluted

NEGATIVE CONTROL
- amount apllied: 500 µL 10% citric acid

POSITIVE CONTROL
- amount applied: one pellet of sodium hydroxide
Duration of treatment / exposure:
- min. 3 minutes, max. 60 minutes (negative control)
Number of replicates:
Four vials were used for the test substance and one vial for the positive control and negative control each.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Duration of treatment / exposure:
min. 3 minutes, max. 60 minutes (negative control)
Details on study design:
ACCEPTANCE CRITERIA
1. breakthrough time for the positive control substance was in the historic control range (mean ± 2-3x standard deviations)
2. negative control was not to induce membrance breakthrough within a 60-minute observation period.
Irritation / corrosion parameter:
penetration time (in minutes)
Value:
7.01
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: breakthrough time of NaOH (solid, historical data) was 12.10 minutes.

Breakthrough times of the test substance, the negative control (NC) and positive control (PC). NB = no breakthrough within maximum observation period (60 minutes).

Test Substance

Break Through Time [min:s]

Vial 1

Vial 2

Vial 3

Vial 4

Mean

8:48

6:49

7:01

5:26

7:01

Controls:

PC: sodium hydroxide, solid

13:27

-

-

-

-

NC: 10% citric acid

NB

-

-

-

-

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-01-17 - 2017-05-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012-07-06
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Name (as cited in the study): Golpanol MPA
- Purity: 99.2 area%
- Batch Nr.: 85603856PO
- Contet: w(C5H9N) = 89.2 g/100 g
- Homogeneity: the test substance was homogeneous by visual inspection
- pH value: ca. 6
- Physical state / color: liquid / colorless, clear
- Storage conditions: room temperature
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
- Test kit: EpiDerm™ 200, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, containing: 24 EPI-200 tissues (reconstructed epidermis); surface 0.6 cm² cultured in Millicells®, diameter 1 cm
- Tissue for MTT reduction control: EPI-200 tissue that is killed by freezing at –20°C
- Assay medium: EPI-100-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)
- 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 /
Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.
- Incubation conditions: 37°C ± 1°C, 5% ± 1%CO2, 90% ± 5% humidity

EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220
- Spectrophotometer: SunriseTM Absorbance Reader for the determination of the optical density of colored extracts (filter wavelength: 570 nm without reference filter)

CONTROLS
Negative control (NC): PBS, sterile
Positive control (PC): 5% (w/v) sodium dodecyl sulfate (SDS) in water
MTT reduction control: PBS, sterile or test substance (irritation test)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- 30 μL undiluted liquid test substance
- Control: 30 μL sterile PBS (negative control NC, NC KC) or with 30 μL 5% SDS (positive control PC) or test substance (killed control KC)
Duration of treatment / exposure:
- 25 minutes at room temperature
- 35 minutes in the incubator
Duration of post-treatment incubation (if applicable):
- First step: 24 +/- 2 hours
- Second step (after medium peplacement): 18 +/- 2 hours
- Step 3: 3 hours
Number of replicates:
- Three tissues each for test substance, negative control, positive control and killed control
Details on study design:
ACCEPTANCE CRITERIA

1) Barrier function and Quality control (QC):
- lower acceptance limit: ET50 = 4.0 hours
- upper acceptance limit: ET50 = 8.7 hours

2) Negative control (NC):
- mean OD_570 of the NC is ≥ 0.8
- mean OD_570 of the NC should not exceed 2.8

3) Positive control (PC):
- viability of ≤ 20%

4) Inter-tissue variability (between all treatments):
- SD of % viability is ≤ 18%

5) Killed controls (KC):
- OD_570 for the KC of the NC should be ≤ 0.35
- OD_570 value for direct MTT reduction of a test substance should be ≤ 30% of the OD570 of the NC
Irritation / corrosion parameter:
% tissue viability
Remarks:
exposure period: 1 h
Value:
2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes

Individual and mean OD570 values, individual and mean viability values and standard deviations (SD).

Exposure period: 1 h

Test substance identification

 

 

tissue 1

tissue 2

tissue 3 

mean

SD

Negative control

viable tissues

mean OD570

1.900

1.797

1.810

1.836

 

viability [% of NC]

103.5

97.9

98.6

100.0

3.1

KC tissues

mean OD570

0.055

0.065

0.052

0.057

 

viability [% of NC]

3.0

3.5

2.8

3.1

0.4

Test substance

viable tissues

mean OD570

0.037

0.036

0.036

0.036

 

viability [% of NC]

2.0

2.0

2.0

2.0

0.0

KC tissues

mean OD570 KC NC corrected

0.000

0.000

0.000

0.000

 

viability [% of NC]

0.0

0.0

0.0

0.0

0.0

Final mean viability of tissues after KC correction

[% of NC]

5.2

2.0

 

Positive control

viable tissues

mean OD570

0.036

0.041

0.036

0.037

 

viability [% of NC]

1.9

2.2

1.9

2.0

0.2

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-01-17 to 2017-05-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Name (as cited in the study): Golpanol MPA
- Purity: 99.2 area%
- Batch Nr.: 85603856PO
- Contet: w(C5H9N) = 89.2 g/100 g
- Homogeneity: the test substance was homogeneous by visual inspection
- pH value: ca. 6
- Physical state / color: liquid / colorless, clear
- Storage conditions: room temperature
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
- Test kit: EpiDerm™ 200, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, containing: 24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells®, diameter 1 cm
- Tissue for MTT reduction control: EPI-200 tissue that is killed by freezing at –20°C
- Assay medium: EPI-100-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)
- 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 /
Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

INCUBATION CONDITIONS
- 37°C ± 1°C, 5% ± 1%CO2, 90% ± 5% humidity

EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220
- Spectrophotometer: SunriseTM Absorbance Reader for the determination of the optical density of colored extracts (filter wavelength: 570 nm without reference filter)

CONTROLS
- Negative control (NC): Deionized water
- Positive control (PC): 8-N potassium hydroxide solution
- MTT reduction control: Deionized water or test substance
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
- 50 μL undiluted liquid test substance
- Control: 50 μL deionized water (negative control NC, NC KC) or with 50 μL 8 N potassium hydroxide (positive control PC) or test substance (KC)
Duration of treatment / exposure:
3 min, 1 h
Number of replicates:
- 2 tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator) and test group (test material, negative control and positive control; 12 tissues per test)
- 2 killed control tissues per exposure time were treated with the test substance and the NC, respectively, to detect direct MTT reduction
Details on study design:
ACCEPTANCE CRITERIA

1) Barrier function and Quality control (QC):
- Lower acceptance limit: ET50 = 4.0 hours
- Upper acceptance limit: ET50 = 8.7 hours

2) Negative control NC:
- mean OD_570 of the NC is ≥ 0.8
- mean OD_570 of the NC should not exceed 2.8

3) Positive control PC:
- tissue viability of ≤ 30% is acceptable for the 3-minute exposure
- mean viability of the tissues exposed for 1 hour should be <15%

4) Variability of the tissues:
- coefficient of variation (CV) of % viability is ≤ 30% (in the range of 20% and 100% viability)

5) Killed controls KC:
- OD_570 for the KC of the NC should be ≤ 0.35
- OD_570 value for direct MTT reduction of a test substance should be ≤ 30% of the OD570 of the NC

Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure period: 3 min
Run / experiment:
1
Value:
78.2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure period: 1 h
Run / experiment:
2
Value:
5.2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
In the first run, the results of the KC indicated an increased MTT reduction (mean viability 0.5 % of NC). Thus, for the test substance the final mean viability is given after KC correction.

Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values, standard deviations (SD) and coefficient variation (CV).

Exposure period: 3 min

Test substance identification

 

 

tissue 1

tissue 2

mean

SD

CV [%]

Negative control

viable tissues

mean OD570

1.527

1.721

1.624

 

 

viability [% of NC]

94.0

106.0

100.0

8.5

8.5

KC tissues

mean OD570

0.085

0.091

0.088

 

 

viability [% of NC]

5.2

5.6

5.4

0.2

 

Test substance

viable tissues

mean OD570

1.195

1.364

1.279

 

 

viability [% of NC]

73.6

84.0

78.8

7.4

9.3

KC tissues

mean OD570 KC NC corrected

0.005

0.012

0.009

 

 

viability [% of NC]

0.3

0.8

0.5

0.3

 

Final mean viability of tissues after KC correction [% of NC]

78.2

 

 

Positive control

viable tissues

mean OD570

0.159

0.244

0.202

 

 

viability [% of NC]

9.8

15.0

12.4

3.7

29.8

Exposure period 1 hour: Individual and mean OD570 values, individual and mean viability values, standard deviations (SD) and coefficient variation (CV).

Exposure period: 1 h

Test substance identification

 

 

tissue 1

tissue 2

mean

SD

CV [%]

Negatvie control

viable tissues

mean OD570

1.659

1.564

1.612

 

 

viability [% of NC]

102.9

97.1

100.0

4.1

4.1

KC tissues

mean OD570

0.101

0.123

0.112

 

 

viability [% of NC]

6.3

7.6

7.0

1.0

 

Test substance

viable tissues

mean OD570

0.086

0.083

0.084

 

 

viability [% of NC]

5.3

5.1

5.2

0.1

2.5

KC tissues

mean OD570 KC NC corrected

0.000

0.000

0.000

 

 

viability [% of NC]

0.0

0.0

0.0

0.3

 

Final mean viability of tissues after KC correction

[% of NC]

5.2

 

 

Positive control

viable tissues

mean OD570

0.097

0.085

0.091

 

 

viability [% of NC]

6.0

5.3

5.6

0.5

9.7

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Remarks:
According to OECD TG 431 a combination of optional Sub-categories 1B-and-1 C
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1979-10-06
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reference:
Composition 0
Qualifier:
no guideline followed
Qualifier:
according to
Guideline:
other: internal method comparabel to OECD TG 404
Principles of method if other than guideline:
Documentation insufficient.
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
- Concentration: 8% and 16% aqueous solution
- Physical state: liquid
Species:
rabbit
Strain:
not specified
Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
not specified
Duration of treatment / exposure:
1, 5, 15 minutes; 20 hours
Observation period:
24 hours
Number of animals:
2
Details on study design:
no details available
Irritation parameter:
other:
Remarks:
See any other information on results including table
Basis:
other:
Remarks:
See any other information on results including table
Time point:
other: See any other information on results including table
Score:
ca.
Reversibility:
other:
Remarks:
See any other information on results including table
Remarks on result:
probability of severe irritation
Remarks:
See any other information on results including table

Table 1: Reported test results.

Body site

Duration of exposure

Findings after 24 hours

Findings after 6 days

back

1 minute

no effects

no effects

back

5 minutes

erythema (doubtful)

back

15 minutes

intense erythema

back

20 hours

slight necrosis / surrounded by an erythematous border

massive necrosis

ear

20 hours

intense redness / blisters / anemic

Table 2: Data reported transferred into the OECD TG 404 scoring system according to GAMER & ROSSBACHER (2002).

Exposure duration

Observation period

24 hours after exposure

8 days after exposure

1 min.

0

0

5 min.

0-1

0

15 min.

1-2

0

20 hours

2 (necrosis)

2 (necrosis)

Interpretation of results:
study cannot be used for classification
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
internal test method, grading and scoring according to Draize (1959)
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
- Purity: assumed to contain 100% active ingredient
- Appearance: clear, colorless liquid, pungent odor
- Date received:1969-03-25
Species:
rabbit
Strain:
other: White New Zealand
Details on test animals and environmental conditions:
- test animals were immobilized in a multiple animal restrainer during exposure
- feed (Purina Rabbit Chow and water) was available ad libitum after exposure
Type of coverage:
occlusive
Preparation of test site:
other: clipped, abraded, intact
Vehicle:
unchanged (no vehicle)
Controls:
not required
not specified
Amount / concentration applied:
0.5 mL
Duration of treatment / exposure:
24 hours
Observation period:
24, 48, and 72 hours after application
Number of animals:
6
Details on study design:
- 24-hour patch test comparable to OECD Guideline 404
- single application of 0.5 mL undiluted test substance on intact (three animals) and abraded skin (three animals)
- trunks of the test animals were wrapped with a rubber dam after application
- test animals were immobilized in a multiple animal restrainer during exposure period
- patches removed after exposure period
- evaluated for gross signs of primary irritation at 24, 48, and 72 hours after application.
- posttreatment: feed (Purina Rabbit Chow and water) was available ad libitum.
- grading and scoring according to index scheme of Draize (1959)
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
animal #6
Time point:
24/48/72 h
Score:
2.67
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Remarks:
Index arbitrary because of blanching effect on abraded sites
Irritant / corrosive response data:
Very slight (one intact site only) to severe erythema noted on abraded and intact sites from 24 hours through 72 hours and slight edema at 24 hours on one intactsite only. Blanching was noted on the abraded sites from 24 hours through 72 hours and dark brown areas observed on exposed sites of two abraded and one intact animal.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
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
Study period:
2017-01-05 to 2017-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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)
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010-12-08
Qualifier:
according to
Guideline:
other: see remarks
Version / remarks:
Schrage A, Kolle SN, Rey Moreno MC, Norman K, Raabe H, Curren R, van Ravenzwaay B, and Landsiedel R (2011). The Bovine Corneal Opacity and Permeability Test in Routine Ocular Irritation Testing and its Improvement within the Limits of OECD Test Guideline 437. ATLA 39: 37–53.
Qualifier:
according to
Guideline:
other: see remarks
Version / remarks:
Kolle SN, Rey Moreno MC, Mayer W, van Cott A, van Ravenzwaay B, Landsiedel R. (2015). The EpiOcular™ Eye Irritation Test is the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations: Correlation Analysis of EpiOcular Eye Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes. ATLA 43: 181-198.
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:
Test was waived.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Name (as cited in the study): Golpanol MPA
- Purity: 99.2 area%
- Content: w(C5H9N) = 89.2 g/100 g
- pH value: ca. 6 (undiluted test substance, determined in the lab prior to start of the GLP study)
- Physical state / color: liquid / colorless, clear
- Storage conditions: room temperature
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Test substance: 750 μL (single topical application of undiluted test substance to the epithelial surface of isolated bovine corneas)
- Negative control (NC): 750 μL of deionized water
- Positive controls (PC 1 /PC 2): 750 μL 100% ethanol / 100% dimethylformamide
Duration of treatment / exposure:
- 10 minutes
Duration of post- treatment incubation (in vitro):
- 2 hours
Number of animals or in vitro replicates:
The test substance, a negative control (NC; deionized water) and positive controls (PC1 / PC2; 100% ethanol / 100% dimethylformamide) were applied to three corneas each.
Details on study design:
TEST SYSTEM
- Test system: isolated bovine cornea
- Source: 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

DATA EVALUATION
- In Vitro Irritancy Score (IVIS)
- Permeability Score
- In Vitro Irritancy score (IVIS)
- Histopathological findings

TREATMENT GROUPS
- Test substance
- Negative control (NC): Deionized water
- Positive control (PC): 100% ethanol (PC1) / 100% dimethylformamide (PC2) for liquid test substances and surfactants
- 100% dimethylformamide was additionally used to increase the historic database of the PC.

ACCEPTANCE CRITERIA
1) PC gives an IVIS that falls within two standard deviations of the current historic mean
2) NC responses should result in opacity and permeability values that are not higher than the established upper limits
3) variability between the corneas treated per test substance should be acceptably low

MATERIALS
- 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 Ca2+ and Mg2+ (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

ANALYSIS
- No analysis of test substance preparation was performed because the test substance was applied undiluted

POST-EXPOSURE TREATMENT
- washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red)
- incubation (2 hours)

INCUBATION CONDITIONS
- Temperature: 32°C

MEASUREMENT OF FINAL CORNEAL OPACITY
- visual observations
- opacitometer

REMARKS
Two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test. However, in case of Golpanol MPA, the results derived with BCOP alone were sufficient for a final assessment. Therefore, further testing in EpiOcular was waived.
Irritation parameter:
cornea opacity score
Value:
38.4
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
in vitro irritation score
Value:
73.6
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
fluorescein leakage
Value:
2.352
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: mean corrected OD490 value
Irritation parameter:
other: histopathological score of irritation (HSI)
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Remarks:
severe findings

Test substance identification

Mean Opacity Value

Mean Permeability Value

Mean In Vitro Irritancy Score

Test Substance

38.4

2.352

73.6

Negative Control

2.4

0.000

2.4

Positive Control 1 (Ethanol)

24.1

0.931

38.0

Positive Control 2 (DMF)

99.6

0.517

107.4

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1969-05-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
internal method
Principles of method if other than guideline:
The potential of acute eye irritation was assessed by a single instillation of 0.1 mL test substance into the conjunctival sac of the left eye of nine rabbits. Three eyes were irrigated with tap water two seconds after application, three after four seconds and three eyes were not irrigated, but held closed for one second. During the test, untreated right eyes served as controls. Body weights were recorded both initially and terminally. Corneal demage was proven on day 7, 10 or 14 with 2.0% sodium fluorescein stain. Observations on gross pathology and systematic toxicology were made 24, 48, and 72 hours and 4, 7, 10, and 14 days following application. The acute eye irritation was graded according to Draize, J. H., from the Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics, Assn. of Food and Drug Offcials of the U. S., Austin, Texas, 1959.
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
- clear, colorless liquid with a pungent odor
- material assumed to contain 100% active ingredient
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
- Body weight range at initiation: 2.0 to 3.1 kg.
- Diet: Purina Rabbit Chow and water available ad libitum
- eyes judged free of irritation and corneal damage (confirmed by sodium fluorescein examination) prior to initiation of study
Vehicle:
unchanged (no vehicle)
Controls:
yes
other: untreated right eyes served as controls.
Amount / concentration applied:
- 0.1 mL
Duration of treatment / exposure:
- two and four seconds
Observation period (in vivo):
14 days
Number of animals or in vitro replicates:
9 individuals
Details on study design:
- Application of test substance: single application into the conjunctival sac of the left eye
- Posttreatment: 3 eyes irrigated with 20 mL tap water two seconds after application; 3 eyes not irrigated but held closed for 2 seconds

Irritation parameter:
other: histopathological observations
Remarks on result:
other: See "Any other information on results incl. tables"

1. Principal Toxic Effects

On day 4, one animal with the eye irrigated after two seconds died. Several animals blinked and preened themselves immediately after application of the test item. Four animals showed moderate weight loss.

2. Gross Pathology

The following gross signs were observed: Blanched conjunctivae, marked conjunctival erythema in one irrigated eye, slight to marked chemosis and discharge, slight to moderate iritis, and slight to marked corneal opacity was noted from day through day 14. On Day 7, Day 10, and Day 14 corneal vascularization was noted in two eyes irrigated after two seconds. Due to extreme irritation and destruction of the eye, four irrigated eyes and the three nonirrigated eyes could not be completely graded by Day 10 or Day 14.

3. Terminal Fluorescein Examination

Corneal damage was shown on day 14 in two eyes irrigated after two seconds, in two eyes irrigated after four seconds and in the three nonirrigated eyes. Staining was not possible in the remaining two irrigated eyes because the animal died or the eye was destructed completely.

Interpretation of results:
study cannot be used for classification
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

In vitro

An in vitro irritation test (BASF SE, 70V0058/16A191, 2017) was conducted under GLP conditions according to OECD TG 439 and EU Method B 46 by a single topical application of 30 μL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). The test was performed with three EpiDerm™ tissues which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. The mean viability of the tissues treated with the test substance was 2.0%. All validity criteria were fulfilled. According to the decision criteria in OECD TG 439, the test substance is to be regarded as irritant.

The potential of the test item to cause dermal corrosion was investigated under GLP conditions according to OECD TG 435 (BASF SE, 70V0058/16A191, 2017) in the in vitro membrane barrier test (CorrostexTM). 500 µL of the undiluted test substance were given to the Corrositex® Biobarrier Membrane by a single topical application. Simultaneously, a positive control (sodium hydroxide, solid) and a negative control (10% citric acid, 500µL) were investigated. Four vials were used for the test substance and one vial for the positive control and negative control each. The exposure duration was between min. 3 minutes and max. 60 minutes (negative control). In result, the mean breakthrough time of the test substance was 7 minutes and 1 second. Therefore, the test substance has to be regarded as corrosive.

A Reconstructed Human Epidermis (RHE) Test under GLP conditions according to OECD TG 431 (BASF SE, 70V0058/16A191, 2017) was performed. Endpoint chosen was the reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT). Two EpiDerm™ tissues per exposure time and test group were treated by a single topical application with 50 µL undiluted test substance (test item and freeze-killed control), with 50 µL deionized water (negative control, freeze-killed control) or with 50 µL 8 N potassium hydroxide (positive control). The mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 78.2%, and it was 5.2% after an exposure period of 1 hour. All validity criteria were fulfilled. Based on these results, the test item is considered to be corrosive according to OECD TG 431. 

In vivo

In an in vivo study (Dow AgroSciences, DR-0385-4500-099, 1969), the primary skin irritation was assessed by a 24-hour patch test comparable to OECD Guideline 404. Three adult albino rabbits of the New Zealand strain were dermally exposed (intact sites) to 0.5 mL undiluted test substance for 72 hours under occlusive conditions (patches were removed after 24 hours of exposure). Grading and scoring of skin irritation was performed following the index scheme of Draize (1959). Several clinical and gross pathological signs were observed. After 24 hours slight edema and from 24 hours through 72 hours severe erythema were noted. In result, the mean Primary Irritation Index for intact sites was 2.25. Under the conditions of the present experiment the test substance was found to be irritant and corrosive when applied to the rabbit skin.

In an additional study with low reliability due to methodological, reporting and assessment deficiencies, the skin irritation potential (BASF, XXI-245, 1972) was investigated according to an internal method. The report describes findings after 24 hours and 8 days. The obtained results indicated a strong irritation potential.

Eye:

In vitro

To assess the eye irritating effect of the test substance, a Bovine Corneal Opacity and Permeability Test (BCOP Test) according to OECD TG 437, EU method B.47 and the methods described in Schrage et al. (2011) and Kolle et al. (2015) was conducted. The epithelial surface of three isolated bovine corneas was exposed to a single topical application of 750 μL undiluted test substance for the duration of 10 minutes at 32 °C. Simultaneously, a negative control (750 μL deionized water) and positive controls (750μL 100% ethanol and 100% dimethylformamide respectively) were investigated with three replicates each. In result, a mean In Vitro Irritancy Score (IVIS) of 73.6 was derived, based on a mean Opacity Score of 38.4. The mean Permeability Value was 2.352. Furthermore, severe histopathological damages occured. All validity criteria were fulfilled. Based on the decision criteria cited in OECD TG 437, the test substance has to be classified as GHS Category 1.

In vivo

In an in vivo study (Dow AgroSciences, DR-0385-4500-099, 1969), the potential of acute eye irritation was assessed by a single instillation of 0.1 mL test substance into the conjunctival sac of the left eye of nine rabbits. Three eyes were irrigated with tap water two seconds after application, three after four seconds and three eyes were not irrigated, but held closed for one second. During the test, untreated right eyes served as controls. The acute eye irritation was graded according to Draize (1959). Several clinical signs were noted during the study period. By staining with fluorescein, corneal damage was shown on day 14 in two eyes irrigated after two seconds, in two eyes irrigated after four seconds as well as in the three non-irrigated eyes. Staining was not possible in the remaining two irrigated eyes because either the animal died or the eye was destructed completely. According to GHS criteria, the test substance has to be regarded as irritating to the eyes (GHS Category 1). 

In an additional study with low reliability due to methodological, reporting and assessment deficiencies, the e

ye irritation potential was investigated according to an internal test method (BASF SE.XXI245, 1972). Reported test results indicated a severe eye irritation potential.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes. Based on available data on skin irritation/corrosion and eye irritation, the test item is classified for skin corrosion GHS Category 1B (H 314: Causes severe skin burns and eye damage) and for eye irritation Category 1 ( H318: Causes serious eye damage) according to Regulation (EC) No 1272/2008 (CLP) as amended for the tenth time in Regulation (EU) No 2017/776.