Methyl 5-mesyl-o-anisate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

Reconstructed Human Epidermis Test Method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 19,2016 - November 4, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

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)

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:

Methyl 5-mesyl-o-anisate

EC Number:

264-267-7

EC Name:

Methyl 5-mesyl-o-anisate

Cas Number:

63484-12-8

Molecular formula:

C10H12O5S

IUPAC Name:

methyl 5-methanesulfonyl-2-methoxybenzoate

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:room temperature

In vitro test system

Test system:

human skin model

Remarks:

SkinEthic RHE® 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:

The SkinEthic™ RHE model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) 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)

Remarks:

The test item was applied as supplied

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:SkinEthic RHE® model
- Tissue batch number(s): 16-RHE-114
- Delivery date: 02 November 2016
Expiration date: November 7, 2016
- Date of initiation of testing: 02 November 2016

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: 2h 58 minutes
- 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: tissue batch: O.D.= 1.2 (CV = 1.3%) specification OD>0.7
Historical control data: mean O.D. range = 0.459 - 1.243
- Barrier function:4.2 h SPECIFICATION 4.0h< ET50<10H
- Morphology: 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 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 exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg of test item (32mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution):

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 and 30 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

ca. 71.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean corrected percent viability of the treated tissues was 71.8 %, versus 2.9% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: A yellow non-homogenous suspension was observed after 3 hours of incubation between 36.9°C and 37.8°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT:
In water: A white non-homogenous suspension was obtained after 3 hours of incubation between 37.1°C and 37.8°C, 5% CO2.
In isopropanol: A white non-homogenous suspension was obtained after 2 hours of incubation at room temperature. Therefore the test item will not interfere with the MTT assay and there is no need to add nonspecific coloration controls to the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals. Summary of proficiency chemicals testing according to OECD 439 criteria included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the standard deviation of the negative control group was 8.5%, the acceptablility criteria is SD ≤ 18%, OD mean of negative control is 1.185 and the acceptability criteria is OD >0.6 x<1.5
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: Summary of results:

	SKIN	OD	Mean OD/disc	Mean OD/product	Viability %	Mean viability %	SD	CRITERIA
Negative control	1	1.091	1.135	1.185	95.8	100.00	8.5
		1.301
		1.118
	2	1.292	1.301		109.8
		1.325
		1.285
	3	1.135	1.118		94.4
		1094
		1.125
Positive control	4	0.034	0.034	0.035	2.9	2.9	0.1	Irritant
		0.035
		0.034
	5	0.038	0.036		3.0
		0.036
		0.034
	6	0.036	0.034		2.9
		0.034
		0.031
Test item PH-15/0504	7	0.806	0.801	0.850	67.6	71.8	3.8	Non irritant
		0.792
		0.805
	8	0.878	0.859		72.5
		0.811
		0.889
	9	0.923	0.890		75.1
		0.886
		0.860

The results were expressed as a viability percentage compared with the negative control

validity %= (ODtest item/ OD negative control )*100

The OD values obtained for each test sample were used to calculate a percentage of viability relative

to the negative control, which was arbitrarily set at 100%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corrected percent viability of the treated tissues was 71.8 %, versus 2.9% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.

Executive summary:

The aim of the study was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). This test was performed according to OECD TG 439 (GLP study). The test item was applied at the dose of 16 mg, to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 42 hours and 30 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.The mean corrected percent viability of the treated tissues was 71.8%, versus 2.9 % in the positive control (5% Sodium Dodecyl Sulfate). The test item has to be considered as Non-irritant to skin, corresponding to UN GHS No Category.