Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Solid / red
stored at room temperature
The test substance was homogeneous by visual inspection.
The test item is not surface-treated or otherwise coated.

Test animals / tissue source

Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
not applicable

The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

To assess the ability of the test material to directly reduce MTT a pretest was performed.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: yes (tissue incubations for positive and negative controls included)
Amount / concentration applied:
0.05 mL
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18h
Number of animals or in vitro replicates:
Two tissue samples were used per group.
Details on study design:
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The
quotient of the values indicates the relative tissue viability.

As the substance showed a potency for direct reduction of MTT by the test substance, the OD570 values were also measured in freeze-killed control tissues (KC) to calculate the mean OD570 of the test substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the
mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

The color of a test substance may interfere with the color density produced by metabolic capacity of the tissue and would falsify the test results when residues of the test substance remain on the tissues after washing and are extracted by the isopropanol.
Due to the pink to red color of the test substance a pretest was performed as follows: the test substance was applied to killed cell tissues, incubated and removed by washing. Thereafter extraction in isopropanol was performed and the OD570 of the extract was determined spectrophotometrically. Based on the pretest it was judged that no color control (CC) was necessary (details are available with the raw data).

Results and discussion

In vitro

Results
Irritation parameter:
other: viability (%)
Run / experiment:
mean of both tissues
Value:
85.3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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

In vivo

Other effects:
Slight red discoloration of the test-substance treated tissues and slight compound residues were observed after the washing procedure.

Any other information on results incl. tables

Table 1: Results

tissue 1 tissue 2 mean Inter-tissue variability (%)
Negative control (water) mean OD570 1.806 1.784 1.795
viability (% of NC) 100.6 99.4 100 1.2
test substance mean OD570 1.535 1.525 1.53
viability (% of NC) 85.5 85 85.3 0.6
Positive control (methyl acetate) mean OD570 0.432 0.486 0.46
viability (% of NC) 24.3 27.1 25.6 2.8

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met