Ethyl salicylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.09.-06.10.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

July, 2015

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:

Ethyl salicylate

EC Number:

204-265-5

EC Name:

Ethyl salicylate

Cas Number:

118-61-6

Molecular formula:

C9H10O3

IUPAC Name:

ethyl 2-hydroxybenzoate

Test material form:

liquid

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 cm
- 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 (about 19 h)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

test item, negative control and positive control: 50 μL each

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

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.: 23737
- 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 after which MTT was extracted for 2-3 hours at room temperature

Data evaluation:
1) The mean OD value of the blank control wells (ODBlk) for each experiment were calculated
2) The mean ODBlk from each mean OD value of the same experiment (blank corrected values) were subtracted
3) The mean value of the two aliquots for each tissue (= corrected test item OD) were calculated
4) The mean value of the two relating tissues for each control and test item (= corrected mean OD) were calculated
For further calculations only the corrected mean negative control OD value was needed
5) The corrected OD value of the negative control corresponds to 100% viability
Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

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

Prediction Model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

Acceptability of the Assay:
The results are acceptable according to MatTek Protocol, 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 freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: tissue viability (%)

Run / experiment:

first replicate

Value:

78.5

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: tissue viability (%)

Run / experiment:

second replicate

Value:

82.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

other: tissue viability (%)

Run / experiment:

mean of two replicates

Value:

80.5

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:

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 43.4%, thus the validity of the test system is ensured.

Any other information on results incl. tables

Results after treatment for 30 minutes with the test item and the controls

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	Mean Rel. Absorbance (% of Negative Control)**
Blank	0.039	0.039	0.039	0
NC	2.11	2.057	2.084	2.045	2.023	101.1	2.2	100
	2.027	2.052	2.04	2.001		98.9
PC	0.943	0.921	0.932	0.893	0.878	44.2	1.6	43.4
	0.908	0.892	0.9	0.862		42.6
Test Item	1.637	1.615	1.626	1.588	1.628	78.5	4	80.5
	1.743	1.671	1.707	1.688		82.5

* Mean of two replicate wells after blank correction

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

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

The assay was performed according to OECD TG 492 and in compliance to GLP.

The colourless test item did not prove to be an MTT reducer in the MTT pre-test and it did not 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).

Irritating effects were not observed following incubation with the test item. 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% (80.5%).

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