Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 March, 2017 - 17 April, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

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:

28 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)

Version / remarks:

28 April 2017

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

February 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

EC Number:

278-188-0

EC Name:

Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

Cas Number:

75314-26-0

Molecular formula:

C14H13O4S . NH4 - C70H60O19S6 . 2NH4

IUPAC Name:

Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

Constituent 2

Reference substance name:

Ammonium sulphate

EC Number:

231-984-1

EC Name:

Ammonium sulphate

Cas Number:

7783-20-2

Molecular formula:

H8N2O4S

IUPAC Name:

Ammonium tetraoxosulfate (VI)

Constituent 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

liquid: viscous

Details on test material:

ca 50% aquaous viscous solution
Batch No: INB3209089
Storage: At room temperature, protected from light
Expiring date: 2018-04-30

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the
EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT
reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the
purposes of distinguishing between skin irritating and
no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection;
Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, 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.

Supplier: SKINETHIC Laboratories
4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.: 18-EKIN-010
Expiry date: 12 March 2018

EpiSkinTMSM 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
an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

EpiSkinTMSM KIT Contents

Units: EpiSkinTMSM 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: EpiSkinTMSM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” for incubations.
(Batch No.: 18 MAIN3 011; Exp. Date: 14 March 2018)
A flask of sterile “Assay Medium” for use in MTT assays.
(Batch No.: 18 ESSC 009; Exp. Date: 14 March 2018)

The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8°C.

Indicator for potential false viability

Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when
the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer),
is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

10 µL test item was applied evenly to the epidermal surface of each of the three test skin units.

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

Three replicates were used for the test item and controls, respectively.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

92

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:

2

Value:

122

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:

3

Value:

116

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

Remarks:

mean

Run / experiment:

1-3

Value:

110

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 5

Irritation / corrosion parameter:

other: mean relative viability%

Run / experiment:

1-3

Value:

82

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:

Validity of the Test

The mean OD value of the three negative control tissues was 0.911. The mean OD value obtained for the positive control was 0.062 and this result
corresponds to 7 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the %
viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Indicator for potential false viability

Possible direct MTT reduction with test item:
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data
calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test item:
As the test item has an intrinsic colour (brown), two additional test item-treated tissues were used for the non-specific OD evaluation. The mean OD
(measured at 570 nm) of these tissues was determined as 0.257. The Non Specific Colour % (NSC %) was calculated as 28.2%. Therefore additional data
calculation was necessary.

Cell viability

The results of the optical density (OD) measured at 570 nm of each replicate .

Any other information on results incl. tables

OD values and viability percentages of the controls and test item:

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.898	99
	2	0.920	101
	3	0.915	100
	mean	0.911	100
	standard deviation (SD)		1.28
Positive Control: SDS (5 % aq.)	1	0.066	7
	2	0.043	5
	3	0.077	8
	mean	0.062	7
	standard deviation (SD)		1.91

 

OD values and viability percentages of the test item (including corrected values):

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%)	1	0.842	0.585	92	64
	2	1.107	0.850	122	93
	3	1.056	0.799	116	88
	mean	1.002	0.745	110	82
	standard deviation (SD)			15.43	15.43

OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) (test item treated tissueswithout MTT incubation)	1	0.179	28.2
	2	0.335
	mean	0.257

Remark:TOD_TT:true MTT metabolic conversion

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

EpiSkin^TMSM test of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439,28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes
(± 0.5 min)at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (±1 hour) in an incubator with 5±1% CO₂. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1°C in 5±1% CO₂protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item has an intrinsic colour (brown), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (=) to 50%

of the negative control.

In thisin vitroskin irritation test using the EPISKIN model, the test item Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) did not show significantly reduced cell viability in comparison to the negative control

(mean relative value: 82 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

 

The results obtained from thisin vitroskin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test itemBenzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%)is considered to be non-irritant to skin and is therefore not classified(UN GHS No Category).