Isopentyl salicylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-July 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

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isopentyl salicylate

EC Number:

201-730-4

EC Name:

Isopentyl salicylate

Cas Number:

87-20-7

Molecular formula:

C12H16O3

IUPAC Name:

isopentyl salicylate

Test animals / tissue source

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- EpiOcularTM tissue: normal, human-derived epidermal keratinocytes cultured to form a stratified squamous epithelium
- Surface: 0.6 cm2
- Shipment: at 2 - 8 °C on medium-supplemented agarose gels
- Equilibration step: 15 minutes at room temperature
- Inspection: each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Pre-incubation: standard culture conditions for 1 hour and after refreshing the Assay Medium at standard culture conditions overnight (21h in the experiment).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Volume: 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
- RhCE tissue construct used, including batch number: EpiOcular Kit Lot No.: 23714
- Conditions of exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Washing: extensively rinsing the tissues with Ca++Mg++-free DPBS
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm (OD570), Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1. No reference wavelength measurement was used.
- MTT assay: incubation with 0.3 mL of MTT solution for 180 minutes at standard culture conditions, after incubation with MTT the inserts were incubated with isopropanol at 2-8°C overnight and then shaken for 3 hours at room temperature
- Data evaluation: the following was calculated:
mean of the blank control wells (ODBlk), ODBlk from each OD value of the same experiment (blank corrected values), mean of the two aliquots for each tissue (= corrected test item OD), mean of the two relating tissues for each control and test item (= corrected mean OD) (for further calculations only the corrected mean negative control OD value was needed), corrected OD value of the negative control corresponding to 100% viability (corrected negative control OD = Negative Control OD - ODBlk = 100% Viability)
- Description of evaluation criteria: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is non-irritant and if the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant. A single test composed of at least 2 tissue replicates should be sufficient for a test chemical when the result is unequivocal. In cases of borderline results, such as non- concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests (according to OECD 492).
- Historical data positive control: Mean Viability: 32.0%; Rel. Standard Deviation: 12.8%; Range of Viabilities: 6.90% - 40.4%; Mean Absorption: 0.538; Rel. Standard Deviation: 0.258; Range of Absorbance: 0.107- 0.849
- Historical data negative control: Mean Absorption: 1.65; Rel. Standard Deviation: 0.299; Range of Absorbance: 1.27 – 2.05
- Acceptability of the Assay: The results are acceptable if (1) the negative control OD is > 0.8 and < 2.5, (2) the mean relative viability of the positive control is below 50% of the negative control viability, (3) 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 (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:

in vitro irritation score

Run / experiment:

Single test with two replicates

Value:

94.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The acceptance criteria were met.

Any other information on results incl. tables

Results of the pre-experiment:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item's colour change potential in water or isopropanol did not lead to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue color.

Results of the main experiment:

Results after treatment for 30 minutes

Dose Group	Absorbance Well 1 (Tissue 1/2)	Absorbance Well 2 (Tissue 1/2)	Mean Absorbance* (Tissue 1/2)	Mean Absorbance * Tissue 1 and 2	Mean Absorbance of 2 Tissues*	Rel. Absorbance [%] Tissue 1 and 2**	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Rel. Absorbance [% of Negative Control]**
Blank	0.039	0.040	0.039	0.000				100.0
Negative Control	2.160	2.155	2.158	2.118	2.066	102.5	5.1	100.0
	2.049	2.056	2.053	2.013		97.5
Positive Control	0.579	0.574	0.577	0.538	0.721	26.0	17.7	34.9
	0.955	0.931	0.943	0.904		43.7
Test Item	1.966	1.931	1.949	1.910	1.945	92.4	3.4	94.1
	2.014	2.025	2.019	1.980		95.8

* Mean of two replicate wells after blank correction


** Relative absorbance [rounded values]: 100 x (absorbance test item / positive control) / absorbance negative control

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 76.0% (threshold for irritancy: <= 60%).

Therefore, the substance was found to be not irritant to the eye.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not eye irritant.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of tes item by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes. After treatment with the negative control the absorbance values were well within the required

acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (94.1%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, test item does not possess any eye irritating potential.