Isobutyl salicylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.04.-12.05.2017

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:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Strasse 80, 65189 Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl salicylate

EC Number:

201-729-9

EC Name:

Isobutyl salicylate

Cas Number:

87-19-4

Molecular formula:

C11H14O3

IUPAC Name:

2-methylpropyl 2-hydroxybenzoate

Test animals / tissue source

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- EpiOcular Kit: MatTek Corporation (82105 Bratislava, Slovakia) normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea.
- 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 (nearly 17 hours)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 μL for the test item (undiluted) and the controls

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2

Number of animals or in vitro replicates:

2 replicates (two tissues)

Details on study design:

Details of the test procedure:
- conditions of exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- rinsing the tissues with Ca++Mg++-free DPBS
- MTT assay:
-- After post-treatment incubation of 120 minutes the MTT assay was performed
-- 0.3 mL of MTT solution
-- incubated for 180 minutes at standard culture conditions
-- in 2.0 mL of isopropanol stored overnight at 2-8 °C in the dark (17 hours) and then shaken for 2.5 hours at room temperature
-- The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1)
-- No reference wavelength measurement was used

Data evaluation:
1) The mean OD value of the blank control wells (ODBlk) for each experiment was calculated.
2) The mean value of the two replicates for each tissue was calculated.
3) The mean ODBlk from each mean OD value of the same experiment was subtracted (blank
corrected values).
4) The mean value of the two relating tissues for each control (negative control (NC) and
positive control (PC) and test item (TI) was calculated (ODTI, ODNC, ODPC).
5) The mean OD value of the negative control corresponds to 100% viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

Calculations for Viability Tests only:
1) The percent viability of each of the two relating tissues for each control and test item relative to the negative control (= 100%) was calculated. Viability (%) = 100 x (OD TI OD PC / OD NC) / meanOD NC
2) The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified.
3) The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model

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.
A single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal.
However, 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.

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

Irritation parameter:

other: mean relative cell viability (%)

Run / experiment:

mean of two tissues

Value:

75.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

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 20.6%, thus the validity of the test system is ensured.

In vivo

Other effects:

The main experiment was performed twice. Since in the 1st main experiment the acceptance criteria were not met, a 2nd main experiment was performed. Only the results of the 2nd main experiment are reported.

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	0.038	0.038	0.038	0.000
NC	1.813	1.937	1.875	1.837	1.864	99.5	1.0	100.0
	1.893	1.894	1.894	1.855		100.5
PC	0.424	0.453	0.439	0.401	0.379	21.7	2.3	20.6
	0.395	0.398	0.396	0.358		19.4
Test Item	1.374	1.48	1.427	1.389	1.402	75.2	1.4	75.9
	1.458	1.446	1.452	1.414		76.6

* Mean of two replicate wells after blank correction

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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 study was performed in accordance to OECD TG 492 and in compliance to GLP.

The test item did not prove to be an MTT reducer in the MTT pre-test, 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 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% (75.9%).

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.