Dodecyl nonan-1-oate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 July - 30 Jan 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

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:

9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Dodecyl nonan-1-oate

EC Number:

241-644-4

EC Name:

Dodecyl nonan-1-oate

Cas Number:

17671-26-0

Molecular formula:

C21H42O2

IUPAC Name:

dodecyl nonanoate

Test animals / tissue source

Species:

human

Strain:

other: EpiOcularTM (OCL-200-EIT)

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model. The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013. It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcularTM EIT is intended to differentiate substances that require “no classification" as eye irritant from those that require labelling as either GHS category 1 or 2 for serious eye damage respective eye irritation potential.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells, which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL®, 10 mm Ø). Analysis for tissue functionality and for potential contaminants was passed.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration: undiluted

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 min

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used :
EpiOcularTM tissue model was used. After the assessment and exclusion of direct MTT reduction and colouring potential of the test substance, the well plates were prepared and the tissues were pre-incubated in warm medium under standard culture conditions (37 ± 1.5 °C, 5 ± 0.5 % CO2 and 95 % relative humidity). After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer (Ca2+ Mg2+ free) and then incubated at standard culture conditions for 30 min. 50 μL of the controls and the neat test substance were then applied and incubated for 30 min at standard culture conditions. After exposure the tissue constructs were thoroughly rinsed and transferred to fresh medium for an immersion incubation at room temperature. Afterwards, the tissues were transferred to fresh medium and incubated for 120 min at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

- RhCE tissue construct used, including batch number :
Keratinocyte strain: 4F1188, Lot Number: 27002

- Doses of test chemical and control substances used :
50 μL of undiluted test substance, 50 μL of demineralised water (negative control), 50 μL of methyl acetate (positive control)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
30 min at 37.0 ± 1.5 °C, 12 min immersion incubation at room temperature, 120 min at 37 ± 1.5 °C post-exposure incubation

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

- Wavelength used for quantifying MTT formazan : 570 nm

- Description of the method used to quantify MTT formazan : The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with a plate reader (Versamax® Molecular Devices, Ismaning, Germany). Then the mean OD of the blank isopropanol (OD Blk) was calculated, followed by the subtraction of mean OD Blk of each value of the same experiment (corrected values).
The mean OD of the two replicates for each tissue the mean OD of the two relating tissues for controls and test substance were then calculated.

To calculate the relative absorbance, the following equation was used: viability (%) = (OD corrected of test substance or positive control/ OD corrected of mean negative control)*100

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :

The in vitro eye irritation test is considered valid if it meets the following criteria:
a) mean OD570 of the negative control: > 0.8 and < 2.5
b) mean relative tissue viability of the positive control: < 50% relative to the negative control.
c) variation within replicates: <20%

If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant (no Category according to UN GHS).
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant (Category 2 or Category 1 according to UN GHS; no differentiation between the categories possible).

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : negative contrtol range: 1.27 - 2.16; positive control range: 6.9 - 43.4%

- Complete supporting information for the specific RhCE tissue construct used :
Tissue viability: 1.269 ± 0.039 (accepted range: 1.1 - 3.0)
Barrier function: ET-50: 24.36 min (accepted range: 12.2 - 37.5)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: mean viability (%)

Run / experiment:

mean value of test substance

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: mean viability (%)

Run / experiment:

mean value of positive control

Value:

36.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, but not in this report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD = 1.691 (> 0.8 and < 2.5)
- Acceptance criteria met for positive control: yes, % viability = 36.5% (< 50%)
- Range of historical values if different from the ones specified in the test guideline: 0.6 - 2.1% (< 20%)

Any other information on results incl. tables

Table 1: Results of MTT assay

	Tissue No.	OD570	Mean (OD570)	Mean (OD570) - blank	Mean tissue viability (% of negative control)
Negative control	1	1.674	1.714	1.676	99.1	100
		1.753
	2	1.735	1.744	1.706	100.9
		1.753
Positive control	1	0.626	0.660	0.622	36.8	36.5
		0.693
	2	0.647	0.649	0.611	36.2
		0.652
Test substance	1	1.614	1.626	1.588	93.9	95.0
		1.638
	2	1.676	1.662	1.624	96.1
		1.649

 OD = optical density

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