Methyl salicylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 03 April 2019 to 11 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 491 (Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

MTT solution prepared in Eagle’s MEM without supplements. During the establishment of the STE test, more stable and comparable results were obtained using the medium without supplements. STE test proficiency study was conducted under the same conditions.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl salicylate

EC Number:

204-317-7

EC Name:

Methyl salicylate

Cas Number:

119-36-8

Molecular formula:

C8H8O3

IUPAC Name:

methyl salicylate

Test material form:

liquid

Details on test material:

see "confidential details on test material"

Test animals / tissue source

Species:

other: rabbit corneal cell line SIRC (Statens Seruminstitut Rabbit Cornea)

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
The cytotoxic effect of test items on corneal epithelia cells is an important mode of action leading to corneal epithelium damage and eye irritation. Cell viability in the STE test method is assessed by the quantitative measurement, after extraction from cells, of blue formazan salt produced by the living cells by enzymatic conversion of the vital dye MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], also known as Thiazolyl Blue Tetrazolium Bromide. It was verified that the test substance falls into the applicability domain of the guideline method.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The rabbit corneal cell line SIRC (Statens Seruminstitut Rabbit Cornea) is the cell line that is recommended in the OECD test guideline No 491 and was used for performing the STE test method. SIRCs are growing as confluent monolayers.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

other: Medium Control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): not applicable
- Concentration (if solution): 0.5% (v/v) and 0.05% (v/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): the test item was prepared in physiological saline (0.9% (w/v) NaCl in deionised water) to reach a final concentration of 5% (w/w). Following, this solution was diluted by serial 10-fold dilution with the respective solvent to reach final concentrations of 0.5% (v/v) and 0.05% (v/v).
- Concentration (if solution): /
- Lot/batch no. (if required): /
- Purity: /

Duration of treatment / exposure:

5 minutes at room temperature.

Observation period (in vivo):

not applicable (in vitro).

Duration of post- treatment incubation (in vitro):

Immediately after exposure, cells were washed and MTT cell viability measurement was performed.

Number of animals or in vitro replicates:

All dose groups were tested in three replicate wells.

Details on study design:

- Cell line used, its source, passage number and confluence of cells used for testing :
Cell line: The rabbit corneal cell line SIRC (Statens Seruminstitut Rabbit Cornea)
Source: ATCC
Passage number: The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Cells were propagated 2 to 3 passages in a culture flask before being employed for testing and did not exceed 25 passages from thawing. The cells used for the first experiment were seeded in the first passage after thawing instead of second or third passage.
Seeding of the culture and confluence: exponentially growing stock cultures more than 50% confluent were rinsed with PBS and treated with Trypsin at 37 ± 1.5 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete medium and a single cell suspension was prepared.
Individual wells of a 96-well tissue-culture microtiter plate were inoculated with 0.2 mL complete medium containing approximately 3 x 104 cells/mL (6000 cells per well) in case that the cells were seeded four days prior to the treatment and 1.5 x 104 cells/mL (3000 cells per well) in case that the cells were seeded 5 days prior to the treatment. The seeding day and the day of treatment are included in the calculation of the days for the cell cultivation: e.g. seeding on Friday of 6000 cells/well and treatment on Monday (four days) or seeding on Friday of 3000 cells/well and treatment on Tuesday (five days). The plates were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Cells reached a confluence of more than 80% at the time of testing.
- Number of repetitions and replicates used : 1 repetition and 3 replicates
- Test chemical concentrations used (if different than the ones recommended) : not applicable
- Justification for choice of solvent for each test chemical : solvent recommended in the guideline.
- Duration of exposure to the test chemical (if different than the one recommended) : not applicable
- Description of any modifications of the test procedure : The MTT solution is prepared in Eagle’s minimum essential medium (MEM) without supplements. During the establishment of the STE test at Envigo CRS GmbH, more stable and comparable results between the three independent experiments were obtained using the medium without supplements. The STE test proficiency study was conducted under the same conditions.
- Description of evaluation and decision criteria used :
The optical density (OD) value obtained from the test item, medium and positive control were used to calculate cell viability relative to the solvent control, which is set at 100%. The relative cell viability is expressed as a percentage and obtained by dividing the OD of the test groups (test item, medium or positive controls) by the OD of the respective solvent control after subtracting the OD of blank from both values. For the cell viability calculation of the medium control physiological saline was used as solvent control.
Cell viability [%] = (mean OD test group - mean OD blank) / (mean OD solvent control - mean OD blank) x 100
Similarly, the relative cell viability of each solvent control is expressed as a percentage and obtained by dividing the OD of each solvent control by the OD of the medium control after subtracting the OD of blank from both values. The arithmetic mean of the three wells of the test item, positive and solvent control in each independent experiment was used to calculate the final arithmetic mean of relative cell viability, respectively.
- Reference to historical positive control mean and Standard Deviation (SD) : not reported
- Demonstration of proficiency of the laboratory in performing the test method (e.g. by testing of proficiency substances) or demonstration of reproducible performance of the test method over time: not reported

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: cell viability (%)

Remarks:

Test Item at 0.05%

Run / experiment:

1

Value:

11.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability ≤ 70 %

Results 2

Irritation parameter:

other: cell viability (%)

Remarks:

Test Item at 5%

Run / experiment:

1

Value:

25.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell Viability ≤ 70 %

Results 3

Irritation parameter:

other: cell viability (%)

Remarks:

Test Item 0.05%

Run / experiment:

2

Value:

3.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability ≤ 70 %

Results 4

Irritation parameter:

other: cell viability (%)

Remarks:

Test Item at 5%

Run / experiment:

2

Value:

26.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability ≤ 70 %

Results 5

Irritation parameter:

other: cell viability (%)

Remarks:

Test Item at 0.05%

Run / experiment:

3

Value:

19.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability ≤ 70 %

Results 6

Irritation parameter:

other: cell viability (%)

Remarks:

Test Item at 5%

Run / experiment:

3

Value:

31

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability ≤ 70 %

Results 7

Irritation parameter:

other: mean cell viability (%)

Remarks:

Test Item at 0.05%

Run / experiment:

Mean of 3 runs

Value:

11.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability ≤ 70 %

Results 8

Irritation parameter:

other: mean cell viability (%)

Remarks:

Test Item at 5%

Run / experiment:

Mean of 3 runs

Value:

27.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability ≤ 70 %

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: microtiter well surfaces were observed in the wells of the 5% test item treated cells (possibly reactivity of the test item with plastic of the microtiter plate). This could have led to a higher absorption for the 5% test item treated cells.

ACCEPTANCE OF RESULTS: The acceptance criteria were met in all three independent tests of the test item.

Any other information on results incl. tables

Tables 1. Summary of Results of METHYL SALICYLATE and the controls

Test Group	Cell Viability [%] per Test			Mean Cell Viability [%]	Standard Deviation [%]	Predicted Eye Irritation Potential
	Test 1	Test 2	Test 3
Medium Control	109.8	97.7	115.2	107.6	9.0
Solvent Control (0.9% NaCl)	100.0	100.0	100.0	100.0	0.0
Positive Control	34.9	19.6	13.7	22.7	10.9
Test Item 0.05%	11.6	3.9	19.9	11.8	8.0	Category1
Test Item 5%	25.5	26.8	31.0	27.8	2.9
Solvent control (0.9% NaCl) compared to medium control	91.1	102.4	86.8

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item Methyl Salicylate induced a decrease of the cell viability below 70%. Thus, the test item is classified as “Category 1” for eye irritation or serious eye damage according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.

Executive summary:

This GLP compliant study was performed to assess the eye irritation potential of the test item Methyl Salicylate in vitro vivo using the rabbit corneal cell line SIRC. This test was performed according to the OECD Test Guideline No. 491 (25 June 2018).

Material and methods

Solutions of the test item with concentrations of 0.05 and 5% were prepared using physiological saline. Both concentrations (0.05 and 5%) of the test item were tested three times with three replicates per test. The cells were incubated for 5 minutes at room temperature.

Furthermore, complete medium was used as medium control. The solvent control for the test item was physiological saline. A 0.01% solution of SLS in physiological saline was used as positive control.

Results

Complete medium did not induce cytotoxic effects in all the tests. The positive control induced an expected distinct reduction in cell viability in all tests within the range of the acceptance criteria.

The medium control showed an OD of ≥ 0.3 after subtraction of blank OD in all tests.

The acceptance criteria were met in all three independent tests of the test item.

Toxic effects were observed following incubation with the two tested concentrations of 0.05% and 5% in all of the runs. The cell viabilities were reduced below 70% for the 0.05% test item treated cells in the range between 3.9% and 19.9% and for the 5% test item treated cells in the range between 25.5% and 31.0%. The slightly higher measured cell viability of the 5% test item treated cells in comparison to the cell viability of the 0.05% treated cells was possibly due to changes of the microtiter well surfaces only observed in the wells of the 5% test item treated cells (possibly reactivity of the test item with plastic of the microtiter plate). This could have led to a higher absorption for the 5% test item treated cells. However, since both test item concentrations induced a decrease of the cell viability below 70%, the test item Methyl Salicylate is classified as “Category 1” for eye irritation or serious eye damage according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.

Conclusion

Under the experimental conditions of this study, the test item Methyl Salicylate induced a decrease of the cell viability below 70%. Thus, the test item is classified as “Category 1” for eye irritation or serious eye damage according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.