1,2-Ethandiylbis(oxy-2,1-ethandiyl)-dimethansulfonat

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 18 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40Bis: "In Vitro Skin Corrosion: H uman Skin Model Test"

Version / remarks:

Official Journal of the European Union No. L142, amended by Commission Regulation (EU) 2019/1390 of 31 July 2019

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: In Vitro EpiDermTM Skin Corrosion Test, For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm

Version / remarks:

27 July 2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

Cas Number:

80322-82-3

Molecular formula:

C8H18O8S2

IUPAC Name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

1,2-Ethandiylbis(oxy-2,1-ethandiyl)-monomethanesulfonat

IUPAC Name:

1,2-Ethandiylbis(oxy-2,1-ethandiyl)-monomethanesulfonat

Test material form:

liquid

Details on test material:

Appearance: Dark yellow liquid
Batch No.: 01110008
Storage: -5 – 40 °C, container tightly closed, protected from light

Specific details on test material used for the study:

Batch No.: 01110008
Storage: At room temperature (max 40 °C), protected from light

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™ (EPI-200, MatTek, Ashland, MA, USA and Bratislava, Slovakia)

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In OECD Test Guideline No. 431 EpiDerm™ Skin Corrosivity Test (SCT) (EPI-200) is referred to as a Validated Reference Method that – based on prevalidation studies, followed by a formal validation study for assessing skin corrosion – could be used for regulatory purposes.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Reconstructed Human Epidermis (RhE) Tissue
- Model used: EpiDerm™ (EPI-200, MatTek, Ashland, MA, USA and Bratislava, Slovakia)
- Supplier: MatTek In Vitro Life Science Laboratories Mlynské Nivy 73, 821 05 Bratislava, Slovakia
- Preparation of EpiDerm™ tissues: 16 March 2022

Standard Assay Kit Components
- Sealed 24-well and 12-well EpiDerm™ (EPI-200-SIT and EPI-212-SIT) plate
* Supplier: MatTek
* Lot Number: 36129
* Storage condition: Refrigerator (5±3 °C)
* Expiry date: 18 March 2022

- Assay medium (EPI-100-ASY)
* Supplier: MatTek
* Lot Number: 031022MJB
* Storage condition: Refrigerator (5±3 °C)
* Expiry date: 24 March 2022

PREPARATION OF EpiDerm™ TISSUE FOR TREATMENT
After the reception procedure of the test kit, the following steps were conducted before the treatment:
- After the medium has reached room temperature (the bottle of medium was placed for approximately 20 minutes to room temperature), the appropriate number of assay plate wells (6 well plates) were filled with the medium (0.9 mL per each well).
- Under sterile conditions, each insert, containing the epidermal tissue was taken out and any remaining agarose that adhered to the outer sides of the insert was removed by gentle blotting on the sterile filter paper or gauze.
- The quality of each insert was checked (unaided eye assessment). Neither tissue defect nor excess moisture on the surface was recorded.
- After the quality check, the surface of the tissues was dried with a sterile cotton tip swab and thereafter the inserts were transferred into the 6-well plates (pre-filled with 0.9 mL medium, upper row) and pre-incubated for 60 minutes at 37±1 °C in an incubator with 5±1 % CO2, 95±1 % humidified atmosphere.
- After incubation of 60 minutes, every tissue was transferred into the wells of the lower rows of the 6-well plates (pre-filled with 0.9 mL medium) and all 6-well plates were incubated at standard culture conditions (37±1 °C, 5±1 % CO2, 95±1 % humidified atmosphere) overnight.

APPLICATION/EXPOSURE
- Preparation of the holding plate: The appropriate number of assay plate wells (24-well plate / each exposure time) were filled with the assay medium (300 µL per well).
- Preparation for the application: After the overnight incubation, but before the application the integrity of the tissues was checked. After the quality check, the surface of the tissues was dried with a sterile cotton tip swab. Two replicates were used for the test item and each control, respectively in each exposure time.
- Test Item: A volume of 50 µL test item was applied evenly to the epidermal surface of each of the two test skin units. The test item was spread gently with the pipette tip in order to evenly cover all of the epidermal surface. Application of the nylon mesh was not necessary.
- Positive and negative control: A volume of 50 µL positive control (8N KOH solution) or negative control (sterile deionized water) were applied on the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface. Application of the nylon mesh was not necessary.
- Additional controls for dyes and chemicals able to colour the tissue: In addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving) in each exposure time.
- One hour exposure: The plates with the treated epidermis units were incubated for the exposure time of 60 minutes (+ 3 min) at standard culture conditions (37±1 °C, 5±1 % CO2, 95 % humidified atmosphere).
- 3 minutes exposure: The plates with the treated epidermis units were incubated for the exposure time of 3 minutes at room temperature. Each control was tested with every exposure time (60 and 3 minutes).

REMOVAL OF TEST MATERIAL
After the exposure time the EpiDermTM units were removed and rinsed thoroughly with 1x DPBS sterile solution to remove all of the test material from the epidermal surface. The constant soft stream of DPBS was used from approximately 1.5 cm distance, and filling and emptying the tissue insert was completed 20 times. The rest of the DPBS was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).
Following rinsing tissues were transferred and kept in the 24-well holding plates (pre-filled with 300 µL medium) until the rinsing procedure was completed. The rest of the DPBS and/or test item was removed from the tissues by gently sweeping the tissues’ surface with a sterile cotton tipped swab (care was taken to avoid the damage of tissues).

MTT TEST
After the exposure of test item was terminated by rinsing with 1xDPBS, the EpiDerm™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL / per well) and then incubated for 3 hours (± 5 min) at 37±1 °C in an incubator with 5±1 % CO2, 95±1 % humidified atmosphere in the dark. An exception were the additional colour controls (NSCliving), whose wells were filled up with 300 µL assay medium (instead of MTT ready to use solution), the other steps remained the same.

FORMAZAN EXTRACTION
After the MTT incubation, the MTT medium was removed (gently aspirated) from all the wells with a Pasteur pipette (or suitable pipette tip) linked to a vacuum source. After removing all the possible amount of MTT medium the wells were refilled with 1xDPBS solution and were aspirated again. This rinsing process with DPBS was repeated twice and after the last aspiration the dryness of the tissue surface was checked and the inserts were transferred into the new 24-well plate. After the transportation, 2 mL extractant solution MTT-100-EXT (MTT-100 Assay Kit from MatTek) was pipetted into each well (extractant solution was flowing into the insert on the tissue surface). The plates were sealed with parafilm and were stored overnight at room temperature in the dark.
After the overnight storage the plates were placed on an orbital plate shaker and shaken (~120 rpm) for 2 hours at room temperature in each exposure time. At the end of the extraction period, the tissue was pierced and the liquid within each insert was allowed to run into the well from which it was taken.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction the extracts were homogenized by pipetting them up and down 3 times, then 2x200 µL samples from each well from the 24-well plate were pipetted into the wells of a 96-well plate. The Absorbance / Optical Density (OD) of the samples was read in the spectrophotometer at the wavelength of 570 nm using MTT-100-EXT solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA
STEP 1
- The test substance is considered to be non-corrosive to skin if mean tissue viability is >= 50 % after 3 min exposure AND >= 15% after 60 min exposure
- The test substance is considered to be corrosive to skin if mean tissue viability is < 50 % after 3 min exposure OR >= 50 % after 3 min exposure AND < 15 % after 60 min exposure
STEP 2 for materials identified as Corrosive in STEP 1
- The test substance is considered corrosive to skin (Sub-category 1A) if mean tissue viability is < 25 % after 3 min exposure
- The test substance is considered corrosive to skin (Sub-category 1B or 1C) if mean tissue viability is >= 25 % after 3 min exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

VEHICLE
No vehicle

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 448 g/L (g KOH/L H2O)

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

60 and 3 minutes for test item, positive control and negative control

Duration of post-treatment incubation (if applicable):

no post-treatment incubation

Number of replicates:

Two replicates were used for the test item and controls, respectively

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 minutes exposure

Run / experiment:

mean

Value:

72

Negative controls validity:

valid

Remarks:

100% viability, SD 3.027

Positive controls validity:

valid

Remarks:

valid 2% viability, SD 0.123

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minutes exposure

Run / experiment:

mean

Value:

99

Negative controls validity:

valid

Remarks:

100% viability, SD 8.203

Positive controls validity:

valid

Remarks:

valid 12% viability, SD 0.625

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

INDICATOR FOR POTENTIAL FALSE VIABILITY
- Possible direct MTT reduction with test item: Colour change was not observed after one hour of incubation. The test item 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: The test item has an intrinsic colour (dark yellow). If the test item is not white, off white, almost white and/or colorless the additional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents from a false viability, which can possibly be caused by the stained surface of the tissues by the test item. Two additional test item-treated tissues were used in each exposure time (60 and 3 minutes) for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was 0.005 and 0.002 at 60 and 3 minutes exposure respectively. The Non Specific Colour living % (NSCliving %) was calculated 0% at each exposure. Both results were under the threshold value (5%), so the correction with the NSCliving % was not necessary. A false estimation of viability can be precluded.

VALIDITY OF THE TEST
- Acceptance criteria met for negative control tissues (OD between 0.8 and 2.8): Yes, mean OD value of the two negative control tissues was 1.746 and 1.568 at 60 and 3 minutes exposure respectively.
- Accpetance criteria met for positive control (mean % viability after 1hr exposure <= 15%): Yes, mean OD value obtained for the positive control was 0.027 at 60 minutes exposure which result corresponds to 2 % viability, when compared to the results obtained from the negative control.
- Acceptance criteria met for variability between tissue replicates (CV <= 30% within the range of 20 - 100% viability): Yes, each calculated Coefficient of Variation value (CV) in the range of 20-100 % viability was below 30.
- Acceptance criteria met for mean OD value of blank samples (<0.1): Yes, mean OD value of the blank samples MTT-100-EXT were 0.0341 and 0.0340 at 60 and 3 minutes exposure (below 0.1).

All validity criteria were within acceptable limits and therefore the study was considered as valid.

Any other information on results incl. tables

Cell viability

 

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

- OD values and viability percentages of the controls:

Controls	Optical Density (OD)		Viability (%)
Negative Control: Sterile deionized water 60 min exposure	1 2	1.709 1.784	98 102
	mean	1.746	100
	SD	/	3.027
	CV	/	3.027
Negative Control: Sterile deionized water 3 min exposure	1 2	1.477 1.658	94 106
	mean	1.568	100
	SD	/	8.203
	CV	/	8.203
Positive Control: Potassium hydroxide (KOH) 8N solution 60 min exposure	1 2	0.028 0.025	2 1
	mean	0.027	2
	SD	/	0.123
	CV	/	8.060
Positive Control: Potassium hydroxide (KOH) 8N solution 3 min exposure	1 2	0.196 0.182	13 12
	mean	0.189	12
	SD	/	0.625
	CV	/	5.176

- OD values and viability percentages of the test item:

Test Item	Optical Density (OD)		Viability (%)
Triethylene glycol dimethanesulfonate 60 min exposure	1 2	1.430 1.101	82 63
	mean	1.265	72
	SD	/	13.323
	CV	/	18.390
Triethylene glycol dimethanesulfonate 3 min exposure	1 2	1.721 1.370	110 87
	mean	1.545	99
	SD	/	15.836
	CV	/	16.065

Mean blank OD values were 0.0341 and 0.0340 at 60 and 3 minutes exposure respectively.

- OD values and NSC_living % of additional controls:

Test Item	Optical Density (OD)		Non Specific Colour % (NSCliving %)
Triethylene glycol dimethanesulfonate 60 min exposure	1 2	0.008 0.002	0 0
	mean	0.005
Triethylene glycol dimethanesulfonate 3 min exposure	1 2	0.001 0.003	0
	mean	0.002

Mean blank OD values were 0.0341 and 0.0340 at 60 and 3 minutes exposure respectively.

 

Applicant's summary and conclusion

Interpretation of results:

other: The test substance can be classified as Non-corrosive. However, skin irritation potential cannot be excluded. The study is used for classification in a weight of evidence approach.

Conclusions:

In conclusion, in this in vitro skin corrosion test in EpiDermTM model (OECD 431) with Triethylene glycol dimethanesulfonate (CAS-No. 80322-82-3), the results indicate that the test item is not corrosive. According to the UN GHS classification system, Triethylene glycol dimethanesulfonate can be classified as “Non-corrosive”.

Executive summary:

Disks of EpiDermTM (two units / chemical / incubation time) were treated with the test item and incubated for 60 minutes (+ 3 min) at standard culture condition (37±1 °C in an incubator with 5±1 % CO2 in a 95 % humidified atmosphere) and for 3 minutes at room temperature. Exposure of test material was terminated by rinsing with 1x DPBS solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a 95 % humidified atmosphere and protected from light. An exception were the additional colour controls (NSCliving), whose wells were filled up with 300 μL assay medium (instead of MTT ready to use solution), the other steps remained the same. The formazan precipitated was then extracted using MTT-100-EXT and quantified spectrophotometrically.

The test item has an intrinsic colour (dark yellow), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving) in each exposure time.

Potassium hydroxide (KOH) 8N solution and sterile deionized water treated (two units / positive and negative control) epidermis were used as positive and negative controls, respectively in the experiment.

 

For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 60 minutes of exposure is above or equal to 15 % and the mean relative viability after 3 minutes of exposure is above or equal to 50 % of the negative control.

 

The test item did not show significantly reduced cell viability in comparison to the negative control after 60 and 3 minutes exposure. The average test item treated tissue mean viability was 72 % at 60 minutes of exposure and the mean viability was 99 % at 3 minutes of exposure.

 

Positive and negative controls showed the expected optical density (OD) in the experiment and cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.

 

In conclusion, in this in vitro skin corrosion test in EpiDermTM model (OECD 431) with Triethylene glycol dimethanesulfonate (CAS-No. 80322-82-3), the results indicate that the test item is not corrosive. According to the UN GHS classification system, Triethylene glycol dimethanesulfonate can be classified as “Non-corrosive”.