Iron cobalt black spinel

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 - 20 July 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:

Iron cobalt black spinel

EC Number:

269-102-2

EC Name:

Iron cobalt black spinel

Cas Number:

68187-50-8

Molecular formula:

CoFe2O4

IUPAC Name:

Iron cobalt spinel

Test material form:

solid

Specific details on test material used for the study:

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

Test animals / tissue source

Species:

other: human-derived, epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

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 (compare Figure 1). 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: 23769 (1st test run) and 23773 (2nd test run)
(Certificates of Analysis see appendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava,
Slovakia

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: By using a sharp spoon, a bulk volume of ca. 50 µL (ca. 21 mg) test material was applied covering the whole tissue surface.

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2 per treatment

Details on study design:

- RhCE tissue construct used, including batch number
EpiOcularTM model (OCL-200), tissue lot: 23769 (1st test run) and 23773 (2nd test run)

- Volume of test chemical and control substances used: 50 µL

- Duration and temperature of exposure: 6 hours at 37°C in the incubator

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
Due to the intense color of the test substance, it was not possible to evaluate whether the test substance is able to directly reduce MTT. Therefore, freeze-killed control tissues (KC) were treated with the test article and the negative control.

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

- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
Measurement using a filter wavelength 570 nm without reference filter (SunriseTM Absorbance Reader)

- Description of the method used to quantify MTT formazan
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 overnight incubation of the tissues in isopropanol at room temperature or by incubation for at least 2 hours on a plate shaker.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The OD570 value measured and corrected for each individual tissue was calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way was calculated.
A chemical is considered as "irritant" if the mean relative tissue viability with a test material is less than or equal to 60%.
Mean tissue viability (% of negative control)
< 55 Irritant
55 - 65 Borderline
> 65 Non-irritant
The “borderline“ evaluation (60 ± 5%) was statistically determined by 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.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
NC: mean OD = 1.749 (0.249 SD)
PC: mean OD = 0.338 (0.118 SD)

- Complete supporting information for the specific RhCE tissue construct used
Sterility: no contamination
1st run:
Tissue Viability: OD[540-570] = 1.404 (0.131)
Barrier fuction: ET-50 = 17.1 min
2nd run:
Tissue Viability: OD[540-570] = 1.28 (0.176)
Barrier function: ET-50 = 18.39 min

- 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

Results 1

Irritation parameter:

other: viablity in %

Run / experiment:

1. Experiment

Value:

78.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

16.1%

Remarks on result:

other: Due to mechanical damage of tissue 2 (viable tissue and KC tissue) during the washing procedure it was excluded from evaluation and the study was repeated.

Results 2

Irritation parameter:

other: mean viablity in %

Run / experiment:

2.Experiment

Value:

78.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

14%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
During the 1st Experiment mechanical damage occured during the washing procedure. In the 2nd Experiment no tissue damage was visible.

Moderate compound residues remained on the test-substance treated tissues after the washing procedure and these tissues were black discolored.

The results of the KC tissues indicate an increased MTT reduction (mean viability 0.4% of NC). Thus, for the test substance the final mean viability is given after KC correction.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria for tissue viabiliy:
The value for inter-tissue variability of the test substance is 20.3% and therefore minimal out of the acceptance range. Since all other quality criteria of the test were met and the viability values of both tissues are well above the cut off for eye irritation this deviation is not considered to adversely affect the result of this study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met