Isobutyl salicylate

Endpoint summary

Description of key information

The substance is not a skin or an eye irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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

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 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

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

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)

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.     

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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)

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.

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.

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.

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)

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

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 does 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 slighlty 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%. Therefore, the test item is not skin irritating.

Eye irritation

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.

Justification for classification or non-classification

The substance does not induce skin irritation effects. According to the Regulation (EC) No. 1272/2008 CLP/EU GHS the substance is not to be classified as skin irritant.

The substance does not possess any eye irritating potential under the experimental conditions reported. According to Regulation (EC) No. 1272/2008 CLP/EU GHS criteria are not met, no classification is required.