[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 - 21 Jan 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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:

adopted in 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics

Molecular formula:

not applicable due to UVCB substance

IUPAC Name:

Fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics

Test material form:

liquid

Test animals / tissue source

Species:

human

Strain:

other: EpiOcular™

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ EIT was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. The EpiOcular™ EIT was endorsed as an in vitro test that can be used to identify those chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS (No Category).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. The EpiOcular™ tissue mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium.
- RhCE tissue or hCE cell construct used, including batch number: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT), Model; 02 October 2017, Lot No. 30692 (Assay Medium Lot No.: 011821ISA)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

30 ± 2 min at 37 °C, 5% CO2 followed by 12 ± 2 min post-exposure immersion

Duration of post- treatment incubation (in vitro):

120 ±15 min at 37 °C, 5% CO2

Number of animals or in vitro replicates:

duplicates for each treatment and control group

Details on study design:

- Details of the test procedure used: The EpiOcular™ Eye Irritation Test (EIT) consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. The test substance was shown to directly reduce MTT in the direct MTT reduction test. Therefore, an additional test with freeze-killed tissues was performed.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm, 10 nm filter band pass, linearity plot provided in report
- Description of the method used to quantify MTT formazan: The absorbance (OD570) was measured with a plate reader (Labtech LT-4500 microplate reader).
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The test substance is considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is > 60% relative to the negative control treated tissue.
- Acceptable variability between tissue replicates for positive and negative controls : The results are acceptable if the negative control OD is > 0.8 and < 2.5 and if the mean relative viability of the positive control is below 50% of the negative control viability.
- Acceptable variability between tissue replicates for the test chemical: The results are acceptable if the difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (test items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Irritation parameter:

percent tissue viability 

Run / experiment:

30 min exposure

Value:

89.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: Since a pre-test for direct MTT reduction was inconclusive due to the intrinsic colour of the test substance, an additional test with freeze-killed was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results.
- Colour interference with MTT: The test substance did not change the colour when mixed with pure isopropanol and thus passed the colour interference pre-test. The use of colour correction tissues was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (value obtained: 2.220). The negative control acceptance criterion was satisfied.
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was < 50% compared to the negative control (value obtained: 42.9%). The positive control acceptance criterion was satisfied.
- Acceptance criteria met for variability between replicate measurements: The difference in viability between the two relating tissues in each treatment group was < 20%. This acceptance criterion was satisfied.

Any other information on results incl. tables

Detailed results

Table 1: Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of duplicate tissues	Individual tissue viability (%)	Relative mean viability (%)	Difference in viability (%)
Negative Control Item	2.279	2.220	102.7	100*	5.4
	2.161		97.3
Positive Control Item	0.894	0.951	40.3	42.9	5.1
	1.007		45.4
Test Item	1.901	1.993	85.6	89.8	8.3
	2.085		93.9

 

OD: Optical Density

*: The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

Under the conditions of the conducted test, the test substance did not present irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.