Methyl toluene-4-sulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2017 to 02 June 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)

Deviations:

yes

Remarks:

Deionised water was used instead of distilled water in the check test (colouring potential of test-item). However, this fact had no impact on the results or integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, No. 431, (29 July 2016) “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”

Deviations:

yes

Remarks:

Deionised water was used instead of distilled water in the check test (colouring potential of test-item). However, this fact had no impact on the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl toluene-4-sulphonate

EC Number:

201-283-5

EC Name:

Methyl toluene-4-sulphonate

Cas Number:

80-48-8

Molecular formula:

C8H10O3S

IUPAC Name:

methyl 4-methylbenzene-1-sulfonate

Test material form:

solid

Details on test material:

Name: PTSM
Chemical name: Methyl toluene-4-sulphonate
CAS number: 80-48-8
Batch/Lot Number: 609271
Description: White to pale yellow solid
Expiry Date: 01 March 2018
Purity: 99.7%
Storage Conditions: Room temperature
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: three-dimensional human epidermis model.

Source strain:

other: Batch No.: 17-EKIN-022

Details on animal used as source of test system:

EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-022, Expiry Date: 05 June 2017) is a three-dimensional human epidermis model. Adult human derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkinTM(SM) Test Kits used in the present study)..

Kit Contents
Units: EPISKINTM(SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKINTM(SM) biopsy punch for easy sampling of epidermis
Medium:
A flask of sterile “Maintenance Medium” (Batch No.: 17 MAIN3 022; Exp. Date: 07 June 2017)
A flask of sterile “Assay Medium” (Batch No.: 17 ESSC 021; Exp. Date: 07 June 2017)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 μL of test item was applied evenly to the epidermal surface of each of two test units.
50 μL of physiological saline was added to each of the two negative control skin units.
50 μL of glacial acetic acid was added to each of the two positive control skin units.

Duration of treatment / exposure:

Single treatment.

Duration of post-treatment incubation (if applicable):

The plates with the treated epidermis units were incubated for 4 hours

Number of replicates:

Two replicates per test item were used.
Two negative controls and two positive controls were also run in this assay.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item-Mean

Value:

96.6

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:

As the test item was colourless and it had no colouring potential, no additional controls were used for the non-specific OD evaluation.
As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

VIABILITY RESULTS
The mean OD value for the test item treated skin samples showed a 96.6% relative viability compared to the negative control.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (0.786).
The two positive control treated tissues showed 0.7% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 9.4%.
The difference of viability between the two negative control tissue samples in the MTT assay was 8.5%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability
		Measured	Blank corrected	(% RV)
Negative Control:	1	0.867	0.820	104.3
Physiological saline	2	0.800	0.753	95.7
(0.9% (w/v) NaCl)	Mean	--	0.786	100.0
Positive Control:	1	0.056	0.009	1.1
Glacial acetic acid:	2	0.050	0.003	0.3
	Mean	--	0.006	0.7
Test Item:	1	0.771	0.724	92.1
PTSM	2	0.842	0.795	101.1
	Mean	--	0.759	96.6

Notes:

1. Mean blank value was 0.047

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

HISTORICAL CONTROL DATA

(updated 13 May 2016)

	Negative control (Physiological saline)	Positive control (Glacial acetic acid)
Minimum optical density (OD)	0.611	0.005
Maximum optical density (OD)	1.516	0.051
Mean optical density (OD)	0.871	0.017
Standard Deviation (SD)	0.164	0.010
Number of cases	81	81

Note: All optical density (OD) value measured are background corrected values (measured at 570 ± 30 min)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with PTSM, the mean cell viability was 96.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN™(SM) model test with PTSM (Batch number: 609271), the results indicate that the test item is non-corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test of PTSM test item was performed in a reconstructed human epidermis model. EPISKIN™(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

 

Disks of EPISKIN™(SM) (two units) were treated with PTSM test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

 

Following exposure with PTSM, the mean cell viability was 96.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

 

In conclusion, in this in vitro EPISKIN™(SM) model test with PTSM (Batch number: 609271), the results indicate that the test item is non-corrosive to the skin.