Octadecyl docosanoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Oct - 01 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary

Version / remarks:

adopted in 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Octadecyl docosanoate

EC Number:

246-115-1

EC Name:

Octadecyl docosanoate

Cas Number:

24271-12-3

Molecular formula:

C40H80O2

IUPAC Name:

octadecyl docosanoate

Test animals / tissue source

Species:

human

Strain:

other: EpiOcular™ (OCL-200-EIT); Keratinocyte strain: 4F1188

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe.
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). It therefore can be used for regulatory purposes as an initial step in the Bottom-Up approach or as one of the last steps in a Top-Down approach. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. All biological components of the used tissue and the kit culture medium have been tested and are free of the presence of viruses, bacteria and mycoplasma. Analysis for tissue functionality and quality was performed and passed.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg (83.3 mg/cm2)

Duration of treatment / exposure:

6 ± 0.25 h

Duration of post- treatment incubation (in vitro):

25 ± 2 h (Post-soak immersion incubation)
18 ± 0.25 h (Post-treatment incubation)

Number of animals or in vitro replicates:

2 replicates

Details on study design:

- Details of the test procedure used
Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye conversion by the EpiOcular™ tissue construct after topical exposure to the test substance. The percent reduction of cell viability in comparison to untreated negative controls is used to predict eye irritation potential.

- RhCE tissue construct used, including batch number
The EpiOcular™ human cell construct (e.g. OCL-200-EIT, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 27015)

- Doses of test chemical and control substances used
50 mg test item; 50 μL positive control (methyl acetate) and 50 μL negative control (destilled water)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
exposure: 6 ± 0.25 h at 37 ± 1 °C
post-exposure immersion (post-soak): 25 ± 2 min at room temperature
post-exposure incubation: 18 ± 0.25 h at 37 ± 1 °C

- Number of tissue replicates used per test chemical and controls
2 replicates each

- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
570 nm; filter band pass of maximum ± 30 nm

- Description of the method used to quantify MTT formazan
Inserts were removed from the 24-well plate after the incubation time in MTT solution (3 h ± 10 min), the bottom of the insert was blotted on absorbent material and transferred to a 6-well "extraction plates" containing 2 mL isopropanol per well. The plate was sealed to inhibit evaporation. MTT was extracted overnight at 2 - 8 °C in the dark or by shaking on an orbital plate shaker for 2 - 3 h at room temperature. After the extraction the inserts were discarded and 200 μL samples (in duplicate) of the extract were transferred into the wells of a 96-well plate and the absorbance/ optical density was measured in a plate spectrophotometer to determine cell viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test substance is predicted to be non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test substance is predicted to be irritant or corrosive.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
Historical positive and negative control results are provided in the report and meet the acceptance criteria.

- Complete supporting information for the specific RhCE tissue construct used
A copy of the certificate of analysis provided by the manufacturer is included in the study report.

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
No information is provided in the report.

Acceptance criteria:
- Mean absolute OD570 nm of negative control is in the range of 0.8 - 2.5.
- Mean relative viability of positive control is < 50%.
- relative tissue viability difference of replicate tissues is < 20%.

Results and discussion

In vitro

Results

Irritation parameter:

other: mean viability (%)

Run / experiment:

test substance (exposure: 6 ± 0.25 h)

Value:

102.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (1.819) of the two negative control tissues is between 0.8 and 2.5.
- Acceptance criteria met for positive control: Yes, as the mean relative viability for the positive control (19.1%) is below 50% of the control viability.
- Acceptable variability between tissue replicates for positive (PC) and negative controls (NC): Yes, as the difference of % viability was 2.3% and 6.3% for the PC and NC, respectively and thus < 20%.
- Acceptable variability between tissue replicates for the test chemical: Yes, as the variability for the test chemical was 7.4%, and thus < 20%.

- Positive and negative control means based on historical data
historical negative control (mean OD ± SD): 1.664 ± 0.311
historical positive control (mean relative viability [%] ± SD): 28.9 ± 12.0

Any other information on results incl. tables

Table 1: Absorbance values and relative viability of the test substance, negative and positive controls

	Negative control		Positive control		Test substance
Tissue No.	1	2	1	2	1	2
OD570(blank-corrected)	1.818	1.737	0.312	0.365	1.851	1.751
	1.847	1.704	0.328	0.356	1.912	1.751
Mean OD570of the duplicates (blank-corrected)	1.833	1.720	0.320	0.360	1.882	1.751
Mean OD570of 2 replicate tissues (blank-corrected) ± SD	1.776 ± 0.07		0.34 ± 0.20		1.816 ± 0.08
Relative tissue viability [%]	103.2	96.8	18.0	20.3	105.9	98.6
Relative tissue viability difference [%]	6.3		2.3		7.4
Mean relative tissue viability [%]	100.0		19.1		102.2

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:

CLP: not classified