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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation, other
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to chapter 13 for the read-across justification.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
08 Mar 2016 to 03 Aug 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
- Name of test substance: Orasol Yellow 157
- Test-substance No.: 16/0017-1
- Source and lot/batch No. of test material: 003-142715
- Purity test date: c 98 area-% (LC)
- Content: 98.4 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no reactivity

OTHER SPECIFICS:
- pH: Ca. 5
- Physical state / color: Solid / brown
Species:
human
Strain:
other: EpiOcularTM model (OCL-200)
Details on test animals or tissues and environmental conditions:
- EpiOcularTM model (OCL-200): A three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium
- Cell course: All cells used to produce EpiOcularTM are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions.In all cases consent was obtained by these institutions from the donor or the donor's legal next of kin, for the use of the tissues or derivatives of the tissue for research purposes.
- Tissue model: OCL-200
- Tissue Lot Number: 23700 (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:
- Amount of test item: 50 µL
- Amount of positive control: 50 µL
- Amount of negative control: 50 µL
Duration of treatment / exposure:
Total exposure time: 6 h
Duration of post- treatment incubation (in vitro):
Postincubation period: 18 h
Number of animals or in vitro replicates:
2 tissue replicates for a single test is sufficient. In case of borderline results a second test is performed. In case of discordant test results between the 2 tests, a third is performed.
Details on study design:
MATERIAL 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
- Wavelength: 570 nm without reference filter
- Assay medium: OCL-200-ASY
- Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT)
- Extracting agent: Isopropanol p.a

TISSUE PREPARATION:
Preincubation: 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 h, the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 h.
Pretreatment: 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.

APPLICATION OF THE TEST ITEM:
Using a sharp spoon, approx. 50 μL of the undiluted test material was applied covering the whole tissue surface. Control tissues were applied with 50 μL of negative and of positive control. After application, the tissues were placed into the incubator until the total exposure time of 6 h.

REMOVAL AND POSTINCUBATION PERIOD:
To remove the test item, the tissues were washed with sterile PBS. Tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates and 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.
The tissues were incubated at standard culture conditions for 18 hours (postincubation period).

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.

EVALUATION CRITERIA:
A chemical is considered as "irritant", if the mean relative tissue viability with a test material is ≤ 60%.
However a borderline range is statistically determined. Therefore, mean percent tissue viability equal to ± 5% of the cut-off value is considered to be discordant.
Irritation parameter:
other: tissue viability (%)
Run / experiment:
1
Value:
115.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: tissue viability (%)
Run / experiment:
2
Value:
100.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: tissue viability (%)
Run / experiment:
mean
Value:
108.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Yellowish discoloration of the test substance-treated tissues was observed after the washing procedure.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Interpretation of results:
GHS criteria not met
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 Nov 1979 to 15 Nov 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: Federal Register, Vol. 38, No. 187, paragraph 1500.42
Version / remarks:
27.09.73 / page 27029
Principles of method if other than guideline:
The eye irritating potential was tested using Vienna White rabbits (Gaukler), five males and one female. The test substance was applied undiluted (11 mg) to the eye. Signs of irritation were noted after 24, 48 and 72 hours.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test substance as cited in report: Neozapon-orange Vers. 1187
- Substance number as cited in report: 79-594

FORM AS APPLIED IN THE TEST
Unchanged
Species:
rabbit
Strain:
Vienna White
Remarks:
Gaukler
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: males, on average 3.04 kg; female, 3.03 kg
- Diet: Ssniff der Firma Intermast
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 11 mg (corresponding to 0.1 mL)
Duration of treatment / exposure:
Not specified
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
6 (5 males, 1 female)
Details on study design:
OBSERVATION TIME POINTS
24, 48 and 72 hours

REMOVAL OF TEST SUBSTANCE
Not specified

SCORING SYSTEM
See 'any other information on materials and methods incl. tables':
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks:
72 hours last observation time point
Irritation parameter:
cornea opacity score
Basis:
animal: #2, #3, #4, #5, #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: #1, #2, #3, #4, #5, #6
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: #1, #3
Time point:
24/48/72 h
Score:
0.66
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
animal: #2, #4
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0.66
Max. score:
3
Reversibility:
not fully reversible within: 72 hours
Remarks:
72 hours last observation time point
Irritation parameter:
conjunctivae score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal: #1, #2, #3, #4, #5, #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The primary irritation reactivity was determined to be 1 = 1/18*19
Interpretation of results:
GHS criteria not met
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27 Mar 1972 to 4 Apr 1972
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The eye irritating potential of the test substance was tested using one male and one female Vienna White rabbit. 50 mg of the test substance was applied unchanged. Signs of irritation were checked after 1 hour, 24 hour and 8 days.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test substance as cited in report: Neozaponorange RE (60-2819)
Species:
rabbit
Strain:
Vienna White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: male 2.4 kg, female 3.05 kg
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg
Duration of treatment / exposure:
Not specified
Observation period (in vivo):
8 days
Number of animals or in vitro replicates:
1 male, 1 female
Details on study design:
OBSERVATION TIME POINTS
1 hour, 24 hours, 8 days
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 8 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Slight redness in combination with red-brown substance residue was observed 1 hour after application in two animals. This slight redness was still observed in one animal 24 hours after application. 8 days after application of the test substance all animals were symptom free.
Interpretation of results:
GHS criteria not met

Data source

Materials and methods

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
EC Number:
943-133-8
Molecular formula:
C32H22CrN10O8.C10-14H21-29NH2 / C34H24CrN8O6.C10-14H21-29NH2 / C33H23CrN9O7.C10-14H21-29NH2
IUPAC Name:
Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Test material form:
solid

Results and discussion

In vitro

Results
Irritation parameter:
other: tissue viability
Run / experiment:
mean
Value:
108.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Result read-across source Cas No. 85029-89-9

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks:
72 hours last observation time point
Remarks on result:
other: Result read-across source CAS No. 84961-40-0
Irritation parameter:
cornea opacity score
Basis:
animal: #2, #3, #4, #5, #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Remarks:
Result read-across source CAS No. 84961-40-0
Irritation parameter:
iris score
Basis:
animal: #1, #2, #3, #4, #5, #6
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Remarks:
Result read-across source CAS No. 84961-40-0
Irritation parameter:
conjunctivae score
Basis:
animal: #1, #3
Time point:
24/48/72 h
Score:
0.66
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Result read-across source CAS No. 84961-40-0
Irritation parameter:
conjunctivae score
Basis:
animal: #2, #4
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Remarks:
Result read-across source CAS No. 84961-40-0
Irritation parameter:
conjunctivae score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0.66
Max. score:
3
Reversibility:
not fully reversible within: 72 hours
Remarks:
72 hours last observation time point
Remarks on result:
other: Result read-across source CAS No. 84961-40-0
Irritation parameter:
conjunctivae score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: Result read-across source CAS No. 84961-40-0
Irritation parameter:
chemosis score
Basis:
animal: #1, #2, #3, #4, #5, #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Remarks:
Result read-across source CAS No. 84961-40-0
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 8 days
Remarks on result:
other: Result read-across source CAS No. 85029-59-0
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Remarks:
Result read-across source CAS No. 85029-59-0

Applicant's summary and conclusion