Reaction mass of isopentyl salicylate and 2-methylbutyl 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:

From May 16th, 2018 to May 31st, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2018

Report date:

2018

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:

July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of Isoamyl salicylate and 2-Methylbutyl salicylate

IUPAC Name:

Reaction Mass of Isoamyl salicylate and 2-Methylbutyl salicylate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

SkinEthic TM RHE/S17 model (Episkin SA, Lyon) has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No.439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic TM RHE/S17 model (Episkin SA, Lyon)
- Tissue batch number(s): 18-RHE-057
- Delivery date: 29 May 2018
- Expiration date: 4 June 2018
- Date of initiation of testing: 16 May 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μl of a MTT solution at 1.0 mg/mL
- Incubation time: 3 h at 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.2 (CV=3.0%) specification OD > 0.7. Historical negative control mean OD range = 0.653 - 1.194
- Barrier function: 4.7 h (Specification 4 h < ET50 < 10 h)
- Morphology: 5.5 cell layers, absence of significant histological abnormalities, well differentiated epidermis (Specification > 4)
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE. no interference

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes of exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes of exposure is greater than 50%.value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, but the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, and the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430:

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL (DPBS –Dutscher - Batch No. 9120318)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

64.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1

Value:

71.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 2

Value:

72.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 3

Value:

48.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The mean percent viability of the treated tissues was 64.1 %, versus 2.3% in the positive control (5% Sodium Dodecyl sulfate). Therefore, the test item is classified as not irritant. Although the percentage of viability of 1/3 of RHE is ≤ 50% (48.4%) the two others had high viability percentages. (71.7% and 72.3%). Since there is just one epidermis that had borderline results, and according to OECD 439 establishing that if he mean viability gives a clear result, (higher than the range of 50 ± 5%), a second run is not necessary.

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, a full demonstration of proficiency was performed with Episkin-RHE/17 model. As SkinEthic RHE® model is very similar to EpiSkin® model, a reduced validation for the use of SkinEthic RHE ®model was performed (Studies HSMI-2012-A', HSMI-2012-B', HSMI-2012-C' and HSMI-2012-D'). Adequate results were obtained for the evaluated chemicals. Summary of proficiency chemicals tested according to OECD 439 criteria included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, OD mean of negative control is 0.822 and the acceptability criteria is OD> 0.4x <1.5
- Acceptance criteria met for positive control: Yes, the mean OD for the positive control was 0.19 while the SD was 0.2. These results are valid according to historical data. Historical OD ranged between 0.009-0.0028 and SD between 0.1-0.6.
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation of the test item tissue replicates was 13.6%, the acceptability criteria is SD ≤ 18%.

Any other information on results incl. tables

Table 1. Individual and average values of OD after 42 minutes exposure

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL 1	0.876	0.871		106.0	100.0	10.1
		0.877
		0.858
	SPL 2	0.695	0.726	0.822	88.4
		0.748
		0.734
	SPL 3	0.850	0.868		105.6
		0.875
		0.878
Positive control	SPL 4	0.020	0.020		2.4	2.3	0.2	Irritant
		0.020
		0.019
	SPL 5	0.020	0.019	0.019	2.3
		0.018
		0.018
	SPL 6	0.016	0.017		2.1
		0.017
		0.017
Test item PH-18/0232	SPL 10	0.602	0.589		71.7	64.1	13.6	Non irritant
		0.589
		0.576
	SPL 11	0.595	0.594	0.527	72.3
		0.595
		0.591
	SPL 12	0.407	0.398		48.4
		0.397
		0.388

# mean of 3 values (triplicate of the same extract)

OD: optival density

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

Acceptability criteria: SD≤18%.

The acceptance criteria were met.

Notes:

- If the viability obtained for the test item is greater than 50%, the test item has to be considered as non irritant.

- If the viability obtained for the test item is less than or equal to 50%, the test item has to be considered as irritant.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria

Conclusions:

The mean corrected percent viability of the treated tissues was 64.1% . Therefore, the test item has to be considered as non-irritant to the skin.

Executive summary:

The evaluation of the possible irritating effects of the test item has been tested after topical application on in vitro human reconstructed epidermis ( SkinEthic TM RHE/S17 model) in accordance with OECD 439 and EU method B.46, under GLP conditions. The test item was applied, as supplied, at a dose of 16 μL to 3 living reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 42 h post-incubation period at 37ºC, 5% CO2. Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Positive and negative control were run in parallel. All acceptability criteria were met. The percent viability of the replicates was 71.7, 72.3 and 48.4%. Although the percentage of viability of 1/3 of RHE is ≤ 50% (48.4%)  the two others had high viability percentages. (71.7% and 72.3%). Since there is just one epidermis that had borderline results, and according to OECD 439 establishing that if the mean viability gives a clear result, (higher than the range of 50 ± 5%), a second run is not necessary. The mean corrected percent viability of the treated tissues was 64.1%, versus 2.3% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item cannot be classified or skin irritation/corrosion according to CLP Regulation no. 1272/2008.