Isobutyl salicylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.09.-10.10.2016

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted July 28, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Mnisterium 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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm TM tissues (Epi-200-SIT Kit)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Tissue batch number(s):23361

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C+/-1.5°C for 35min and at room temperature until the end of the treatment
- Temperature of post-treatment incubation (if applicable):37+/-1°C

CONTROL
- Negative Control: 30 µL DPBS (Gibco) was used as negative control per tissue;
- Positive Control: 30 µL of a 5% SLS solution in deionised water (MatTek) was used a positive control per tissue, freshly prepared prior to the start of the experiment.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues rinsed with DPBS at least 15 times, then submerged in DPBS at least 3 times, then once again rinsed with sterile DPBS from inside and outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versama Molecular Devices, Softmax Pro, version 4.7.1
- Filter: 570 nm
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: test item does not reduce MTT, therefore, additional test was not performed

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
Viability measured using MTT assay
mean tissue viability < 50%; irritant (I), H315 (category 2)
mean tissue viability > 50%; non-irritant (NI)
Amount/concentration applied:undiluted
Duration of treatment / exposure:60 minutes

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30µL undiluted


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30µL undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

92.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: 30µL of the test substance were added to 0.3 mL of deionised water. The mixture was incubated for 60min at 37 +/-1.5°C, 5 +/-0.5% CO2. At the end of the exposure, the presence and the intensity of the staining were evaluated.
The test item did not dye water or did not change colour in presence of water.
- Colour interference with MTT:30µL of the test substance were added to 1 mL of the MTT-solution (1mg/mL) and was incubated for 60min at 37 +/-1.5°C, 5 +/-0.5% CO2. Untreated MTT medium was used as control.
MTT solution did not turn blue/purple in presence of the test item. The test substance was not considered to reduce MTT.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Results after treatment and the controls:

Dose Group	Exposure internal	Tissue No.	Absorbance 570 nm Well 1	Absorbance 570 nm Well 2	Absorbance 570 nm Well 3	Mean Absorbance of 3 wells	Mean Absorbance of three wells blank corrected	Mean Absorbance of 3 tissues after blank correction*	Rel. Absorbance (%) Tissue 1, 2 +3**	Relative Standard Deviation (%)	Mean Rel. Absorbance (% of negative control)***
Blank			0.038	0.038	0.038	0.038	0.000
Negative Control	60 min	1	1.781	1.796	1.789	1.788	1.751	1.814	96.5	7.4	100
		2	1.997	2.036	1.987	2.006	1.969		108.6
		3	1.760	1.756	1.761	1.759	1.721		94.9
Positive Control	60 min	1	0.144	0.141	0.131	0.139	0.101	0.087	5.6	14.1	4.8
		2	0.126	0.120	0.118	0.121	0.083		4.6
		3	0.114	0.123	0.108	0.115	0.077		4.3
Test substance	60 min	1	1.785	1.753	1.756	1.764	1.726	1.685	95.2	7.8	92.9
		2	1.585	1.556	1.589	1.576	1.538		84.8
		3	1.816	1.839	1.831	1.828	1.791		98.7

* Mean of three replicate wells after blank correction

** Relative absorbance per tissue (rounded value)

*** Relative absorbance per treatment group (rounded value)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not irritant to skin according to EU CLP regulation in this study and under the experimental conditions.

Executive summary:

This test was performed according to the OECD 439 guideline "In vitro Skin Irritation: Reconstructed Human Epidermis Test method" and under GLP.

The study was performed to assess the irritation potential of the test substance by means of the Human Skin Model Test.

The test substance did not show colour/MTT interference in the pre-test.

During the test, 30µL of the test item, the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue and spread to match the surface of triplicate tissues.

After 60 minutes of treatment, the test item, the negative control and the positive control were washed off the skin tissues. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following about 70 hours extraction of the colorant from the cells.

The amount of extracted colorant was determined by photometry at 570 nm.

The acceptance criteria were met according to the OECD 439 guideline:

- Mean of negative control absorbance OD ≥ 0.8 and ≤ 2.8 (value between 1.756 and 2.036)

- Relative absorbance of positive control was ≤ 20% (value 4.8%)

- Relative standard deviation of 3 identical replicates ≤ 18% (value for test item: 7.8%, negative control 7.4% and positive control 14.1%)   

The mean relative absorbance value of the test item was slightly reduced to 92.9% in comparison to the relative absorbance value of the negative control after exposure of the skin tissue to the test substance.

This value is above the threshold for irritancy of ≤ 50%.

In conclusion, it can be stated that in this study and under the experimental conditions, the test substance is not irritant to skin according to EU CLP 1272/2008 regulation criteria.