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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-09 to 2017-08-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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:
2015-07-28
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)
EC Number:
916-868-7
Molecular formula:
C32H22CoN10O8.C10-14H21-29NH2 and C32H22CoN10O8.Na
IUPAC Name:
Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: 002-151203, Test-substance No.: 16/0082-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS: Solid / orange, pH ~ 5 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The EpiOcular tissue system is demanded by the OECD Guideline 492.
- 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 diameter) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: ~ 50 µL (bulk volume, ~ 13 mg)

Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcularTM model (OCL-200), Tissue Lot No 23713, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Doses of test chemical and control substances used:
Test substance: 50µL
Negative Control: 50 µL
Positive Control: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods.
Exposure: 6 h at 37 °C in the incubator
post-exposure immersion: 25 min
post -exposure incubation: 18 h at 37 °C in the incubator
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: the test substance was applied to a KC tissue, incubated for 6 hours and removed by washing. Thereafter extraction in isopropanol was performed and the OD570 of the extract was determined spectrophotometrically. Based on the result of the pretest it was judged, that application of color control tissues was not necessary.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: Spectrophotometer, measurement using a filter wavelength 570 nm without reference filter
- Description of the method used to quantify MTT formazan: 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.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
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 (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 criteria 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 %.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: see Any other information on materials and methods, Table 1.
- Analysis for tissue functionality and quality: see Any other information on materials and methods, Table 2.








Results and discussion

In vitro

Results
Irritation parameter:
other: Cell viability [mean, %]
Value:
104.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control:Yes

Any other information on results incl. tables

Table 4: Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance identification

 

Tissue 1

Tissue 2

Mean

Inter-tissue variability [%]

NC

Mean OD570

1.818

1.638

1.728

 

 

Viability [% of NC]

105.2

94.8

100.0

10.4

Test substance

Mean OD570

1.652

1.957

1.805

 

 

Viability [% of NC]

95.6

113.2

104.4

17.6

PC

Mean OD570

0.392

0.364

0.378

 

 

Viability [% of NC]

22.7

21.1

21.8

1.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met