Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(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-)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2017 - 9 June 2017

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:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(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:

916-867-1

Cas Number:

12270-00-7

Molecular formula:

C32H18CoN6O8Na - C32H20CoN8O8Na - C32H22CoN10O8Na

IUPAC Name:

Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(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
- Source and lot/batch No.of test material: 16/137

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

- Description of the cell system used:
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 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

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:

- RhCE tissue construct used, including batch number:
Tissue model: OCL-200
Tissue Lot Number: 23769
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava,Slovakia

- Doses of test chemical and control substances used:
50 µL of NC (de-ionized water) and PC (Neat methyl acetate)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
6 hours exposure at 37°C in the incubator; 18 hours post-exposure at 37°C in the incubator

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled): 2

- Description of the method used to quantify MTT formazan:
The optical density at a wavelength of 570 nm (OD570) of isopropanol extracts was determined spectrophotometrically (Spectrophotometer: SunriseTM Absorbance Reader)

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability Prediction
(% of negative control)
< 55 Irritant
55 - 65 Borderline
> 65 Non-irritant
The „borderline“-evaluation (60 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.

- Positive and negative control means and acceptance ranges based on historical data
Historic Range of NC
OD570 (Feb 2016 - Jan 2017, solids)
Mean OD 1.749 ± 0.249

Historic Range of PC
OD570 (Feb 2016 - Jan 2017, solids)
Mean OD 0.338 ± 0.118

- Acceptable variability between tissue replicates for positive and negative controls:
NC: 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.
PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.

- Acceptable variability between tissue replicates for the test chemical:
Two tissues were treated under the same conditions. A variability between the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability

Run / experiment:

1st experiment

Value:

70

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

16.1%

Other effects / acceptance of results:

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

OTHER EFFECTS
- Orange discoloration and minimal compound residues were observed on all test-substance treated tissues after the washing procedure.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met