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

Endpoint summary

Description of key information

Skin irritation/corrosion:

- EpiDerm™ test for skin corrosion (OECD 431): cell viabilities: 99 % and 72% after 3 min and 60 min exposure. Not corrosive.

- EpiDerm™ test for skin irritation (OECD 439): cell viabilities: 59.2 % and 79.9%. Not Irritant.

 

Eye irritation/ damage:

- Epiocular™ (OECD 492): 7.4 % cell viability, classified as Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1).

- ICE test (OECD 438): overall ICE class thrice I. Not corrosive.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 April - 24 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

14 June 2021

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No 2019/1390 of 31 July 2019 amending, for the purpose of its
adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test
methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), B.46. In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT), For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDermTM (EPI-200-SIT)

Version / remarks:

19 January 2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Test system:

human skin model

Remarks:

Reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, MA, USA and Bratislava, Slovakia)

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm™ model has been validated for irritation testing in an international trial. The validation trial was in accordance with the principles and criteria documented in OECD Guidance Document No. 34 on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment and ECVAM (2007) Performance Standards for applying human skin models to in vitro skin irritation. In 2008, ESAC concluded that the Modified EpiDerm™ SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances. The Modified EpiDerm™ SIT is an in vitro procedure that, depending on information requirements, allows determining the skin irritancy of chemicals as a stand alone replacement test, as a screen, or within a testing strategy in combination with, if appropriate, a weight of evidence approach.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM reconstructed human epidermal model
- Supplier: MatTek In Vitro Life Science Laboratories Mlynské Nivy 73, Bratislava, 821 05 Slovakia
* First experiment:
- Lot No.: 361351
- Expiry date: 22 April 2022
- Date of initiation of testing: April 21, 2022
*Second experiment
- Lot No.: 361522
- Expiry date: 24 June 2022
- Date of initiation of testing: June 22, 2022

PREPARATION OF EpiDerm™ TISSUES FOR TREATMENT
After the reception procedure of the test kit, the following steps were conducted before the treatment:
After the assay medium reached room temperature (the bottle of assay medium was placed for approximately 20 minutes at room temperature) the appropriate number of assay plate wells (6-well plates) were filled with the assay 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. After that, the tissues were placed in the empty, sterile 24-well plate and quality check of each insert was done by unaided eye assessment. Tissue defects and excess moisture on the surface were not observed. 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 (60±5 minutes) at 37±1 °C in an incubator with 5±1 % CO2, 90±10 % humidified atmosphere. After incubation of one hour, every tissue was transferred into the wells of the lower rows of the 6-well plates (pre-filled with 0.9 mL medium). Then, all 6-well plates were incubated at standard culture conditions (37±1 °C, 5±1 % CO2, 90±10 % humidified atmosphere) overnight (18±3 hours).

APPLICATION
- Preparation for the application: The appropriate number of assay plate wells (6-well plates) were filled with the assay medium (0.9 mL per well) in the upper row. After the overnight incubation but before the application the integrity of each tissue was checked. After the quality check, the surface of the tissues was dried with a sterile cotton tip swab. Three replicates were used for the test item and positive and negative controls, respectively.
- Test Item: A volume of 30 µL test item was applied evenly on the epidermal surface of each of the three 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 30 µL positive control (SDS 5 % aq.) or negative control (1x DPBS) 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.
- 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).

EXPOSURE
The total exposure time was 60 minutes, which included two different exposure periods with different incubation conditions. Each plate with the treated epidermis units was incubated for 35 minutes (± 1 min) at standard culture conditions (37±1 °C, 5±1 % CO2, 90±10 % humidified atmosphere). Furthermore, each plate was incubated for 25 minutes (± 1 min) at room temperature.

RINSING
After the exposure time (60±1 min) 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 stream of 1x DPBS was applied from approximately 1.5 cm distance and filling and emptying the tissue insert was completed 15 times. After the 15th rinse from the washing bottle, the insert was completely submerged three times in clean beakers containing a minimum of 150 mL 1x DPBS solution. Finally, the insert was rinsed once from inside and once from outside with 1x DPBS solution. The rest of the 1x DPBS was decanted onto the absorbent material. Remaining 1x 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).

POST-INCUBATION
After rinsing, the inserts were placed into the 6-well plates (pre-filled with 0.9 mL assay medium per well) in the upper row and then incubated for 24 hours (± 2 h) at 37±1 °C in an incubator with 5±1 % CO2, 90±10 % humidified atmosphere. After incubation of 24±2 hours, the lower row of the 6-well plates was pre-filled with fresh assay medium (0.9 mL per well) and then the inserts were transferred from the upper row to the lower row. Finally, the plates were placed back into the incubator and were incubated for 18±2 hours at 37±1 °C in an incubator with 5±1 % CO2, 90±10 % humidified atmosphere.

MTT TEST
After the post-incubation period the EpiDerm™ units were transferred into the 24-well plate filled with MTT ready to use solution (300 µL per well) and then incubated for 3 hours (± 5 min) at 37±1 °C in an incubator with 5±1 % CO2, 90±10 % humidified atmosphere. An exception was the additional colour controls (NSCliving), whosewells 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 1x DPBS solution and were aspirated again. This rinsing process with 1x DPBS was performed 3 times. 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 transfer, 2 mL extractant solution (MTT-100-EXT) was pipetted into each well (extractant solution was flowing into the insert on the tissue surface). The plates were sealed with parafilm and extracted. To extract the formazan the plate was placed on an orbital plate shaker and shaken (~120 rpm) for 2 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted 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 placed 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 (isopropanol) solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA
The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability after 60 minutes exposure and post incubation is less or equal (<=) to 50 % of the negative control. However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion (OECD 431) will be required to decide on its final classification. In case the test item is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (<=) to 50 %, the test item is considered to be irritant to skin in accordance with UN GHS Category 2.

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: 30 µL

NEGATIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: sterile 1x DPBS (Phosphate Buffered Saline)

POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: Sodium Dodecyl Sulphate (SDS) 5% aq. solution

Duration of treatment / exposure:

60±1 min

Duration of post-treatment incubation (if applicable):

42±2 h

Number of replicates:

Three replicates were used for the test item, the negative control and the positive control.
Two replicates were used for the additional colour control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

79.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

59.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Possible direct MTT reduction with test item: No colour change was observed after one hour of incubation. Therefore, the test item was considered not to interact with the MTT, and 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 (dark yellow), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues were 0.007 in the first and 0.005 in the additional experiment respectively. The Non Specific Colour living % (NSCliving %) were calculated as 0.5 % in the first and 0.2 % in the additional experiment and were under the threshold value (5 %) in both cases. Therefore, additional data calculation was not necessary. A false estimation of viability can be precluded.
- Mesh Compatibility: Reaction with the nylon mesh was not detected.

ACCEPTANCE OF RESULTS:
All validity criteria were within acceptable limits and therefore the study is considered as valid.
- First experiment: The mean OD value of the three negative control tissues was 1.432. The mean OD value obtained for the positive control was 0.094 which result corresponds to 6.6 % viability, when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. The mean OD value of the blank samples was 0.0349, which was under the threshold value of 0.1.
- Additional experiment: The mean OD value of the three negative control tissues was 2.145. The mean OD value obtained for the positive control was 0.056 which result corresponds to 2.6 % viability, when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. The mean OD value of the blank samples was 0.0368, which was under the threshold value of 0.1.

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:

 

Result of the first experiment

- OD values and viability percentages of the controls:

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x DPBS	1 2 3	1.462 1.350 1.485	102.1 94.3 103.7
	mean	1.432	100
	standard deviation (SD)		5.02
Positive Control: SDS (5 % aq.)	1 2 3	0.095 0.092 0.095	6.6 6.4 6.6
	mean	0.094	6.6
	standard deviation (SD)		0.12

 

- OD values and viability percentages of the test item:

Substance	Optical Density (OD)		Viability (%)
Test Item: Triethylene glycol dimethanesulfonate	1 2 3	0.956 0.804 0.781	66.8 56.1 54.5
	mean	0.847	59.2
	standard deviation (SD)		6.65

 

- OD values and NSC_living % of additional control:

Additional colour control	Optical Density (OD)		Viability (%)
Triethylene glycol dimethanesulfonate (test item treated tissues without MTT incubation)	1 2	0.006 0.007	0.5
	mean	0.007
	standard deviation (SD)	0.04

Remark:

NSC_living: Non-Specific Colour in living tissues

Mean blank OD value was 0.0349

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

 

Result of the additional experiment

- OD values and viability percentages of the controls:

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x DPBS	1 2 3	2.097 2.105 2.232	97.8 98.1 104.1
	mean	2.145	100
	standard deviation (SD)		3.53
Positive Control: SDS (5 % aq.)	1 2 3	0.050 0.058 0.059	2.3 2.7 2.8
	mean	0.056	2.6
	standard deviation (SD)		0.25

 

- OD values and viability percentages of the test item:

Substance	Optical Density (OD)		Viability (%)
Test Item: Triethylene glycol dimethanesulfonate	1 2 3	1.690 1.721 1.728	78.8 80.3 80.5
	mean	1.713	79.9
	standard deviation (SD)		0.94

 

- OD values and NSC_living % of additional control:

Additional colour control	Optical Density (OD)		Viability (%)
Triethylene glycol dimethanesulfonate (test item treated tissues without MTT incubation)	1 2	0.004 0.006	0.2
	mean	0.005
	standard deviation (SD)	0.06

Remark:

NSC_living: Non-Specific Colour in living tissues

Mean blank OD value was 0.0368

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

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EpiDermTM model (OECD 439), indicate that the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) reveals no skin irritation potential under the utilised testing conditions. The test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) is considered to be non-irritant to skin and is therefore not classified (UN GHS / CLP No Category).

Executive summary:

EpiDerm^TM Model test of Triethylene glycol dimethanesulfonate (CAS 80322-82-3) 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, 14 June 2021.

 

Unexpected colour change of the medium was observed at the end of the post-incubation period in the first experiment. The effect of this deviation is unknown, so despite the results of the first experiment meeting all acceptance criteria and to avoid any false results, an additional experiment was necessary to confirm or reject the results of the first experiment.

 

Disks of EpiDerm^TM (three units) were treated with the test item and incubated for 25 minutes at room temperature and 35 minutes at standard culture conditions (37±1 °C, 5±1 % CO₂, 90±10 % humidified atmosphere). The (60 minutes) exposure of the test item was terminated by rinsing with 1x DPBS solution. Epidermis units were then incubated at 37±1 °C for approximately 24 hours in an incubator with 5±1 % CO₂, 90±10 % humidified atmosphere. After incubation of approximately 24 hours, the tissues were transferred into fresh medium and the incubation process was continued for approximately 18 hours at standard culture conditions. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37±1 °C in 5±1 % CO₂, 90±10 % humidified atmosphere. The precipitated formazan was then extracted using extractant solution MTT-100-EXT (isopropanol) and quantified spectrophotometrically. The procedure described above was applied in both cases.

 

The test item has an intrinsic colour (dark yellow), therefore two additional test item treated tissues were used for the non-specific optical density (OD) evaluation (NSC_living) in the first and in the additional experiment as well.

 

SDS (5 % aq.) and 1× DPBS 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.

 

A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 60 minutes exposure and post incubation is less or equal (≤) to 50 % of the negative control.

 

In this in vitro skin irritation test using the EpiDerm^TM model, the test item Triethylene glycol dimethanesulfonate did not show significantly reduced cell viability in comparison to the negative control in the first (mean relative viability: 59.2 %) and the additional experiment (mean relative viability: 79.9 %). All obtained test item viability results were above 50 %, when compared to the viability values obtained from the negative control in each run. Therefore, the test item was considered to be non-irritant to skin.

 

Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18. The experiment was considered to be valid.

 

The results obtained from this in vitro skin irritation test, using the EpiDerm^TM model (OECD 439), indicate that the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) reveals no skin irritation potential under the utilised testing conditions. The test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) is considered to be non-irritant to skin and is therefore not classified (UN GHS / CLP No Category).

Reference 2

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

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

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

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)

Specific details on test material used for the study:

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

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

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

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.

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.

 

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”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 - 09 June 2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

02 February 2021

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Species:

other: EpiOcular™ human cell construct (MatTek In Vitro Life Science Laboratories)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 minutes (±2 min)

Duration of post- treatment incubation (in vitro):

12 ± 2 minutes (post-soak)
120 ± 15 minutes (post-treatment incubation)

Number of animals or in vitro replicates:

two replicates per test item and controls, respectively

Details on study design:

DETAILS ON TEST SYSTEM
- RhCE tissue construct used: EpiOcular™ (OCL-200-EIT)
- Supplier: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava, Slovakia
- Lot No.: 34969
- Expiry date: 10 June 2022
- Storage: The preparation of tissues for treatment was started after arrival in TOXI-COOP ZRT.’s Laboratory. Therefore, the storage of EpiOcular™ (OCL-200-EIT) units was not necessary. The assay medium supplied with the kits was stored in refrigerator (2-8 °C).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.

DETAILS ON TEST PROCEDURE
- Preparation of EpiOcular Tissues for treatment: After the test kit arrival, the tissues were equilibrated to room temperature for 15 minutes. The Assay Medium was pre-warmed to room temperature. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37±2 °C in an incubator with 5±1 % CO2, >= 95 % humidified atmosphere for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (16 - 24 hours).

- Application: Two replicates were used for the test item and control(s) respectively.
*Pre-Treatment: After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30±2 minutes.
* Test item: After the 30 ± 2 minutes Ca++Mg++Free-DPBS pre-treatment, amount of 50 µL of the test item was applied topically on the EpiOcular™ tissues. The test item was spread gently with the pipette tip in order to evenly cover all the tissue surface.
* Positive and negative control: A volume of 50 µL positive control (Methyl Acetate) or negative control (sterile deionized water) was applied on the tissues surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the EpiOcular™ tissues surface.
* 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).

- Exposure: The plates with the treated tissue units were incubated for the exposure time of 30 minutes at (±2 min) standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, >=95 % humidified atmosphere).

- Rinsing: After the incubation time the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS as follows: Three clean beakers (glass with minimal 150 mL capacity), containing a minimum of 100 mL each of Ca++Mg++ Free-DPBS was used per test item and controls. Each test item and controls utilized a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitated handling and decanting. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps (taking care not to damage the tissues by the forceps). The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the tissues were dipped into the first beaker of DPBS and were swirled in a circular motion in the liquid for approximately 2 seconds and thereafter were lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The tissue was rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid 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).

- Post-Soak and Post-incubation: After rinsing, the tissues were transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 12 ± 2 minutes immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue. At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37 °C) Assay Medium. The tissues were incubated for 120 ± 15 minutes at standard culture conditions (Post-treatment Incubation).

- MTT Test After Post-incubation: After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±2 °C in an incubator with 5±1 % CO2 protected from light, >=95 % humidified atmosphere (for the Non Specific Colourliving % (NSCliving %) tissues, fresh assay medium was used instead of MTT solution).

- Formazan Extraction: Each insert was removed from the 24-well plate after 3 hours ± 15 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 mL of isopropanol in each designated well so that isopropanol was not flowing into the insert on the tissue surface. The plates were sealed with parafilm and were stored overnight at 2-8 °C in the dark. To extract the formazan, the plates were placed on an orbital plate shaker and shaken (~120 rpm) for approximately 2 and a half hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative and positive controls were treated identically.

- Cell Viability Measurements: Following the formazan extraction, 200 µL sample(s) from each tube (2×200 µL) was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Optical Density) of the samples in the 96-well plate spectrophotometer at the wavelength of 570 nm using isopropanol as the blank (8×200 µL).

Irritation parameter:

mean percent tissue viability 

Value:

7.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Indicator of potential false viability
- Possible direct MTT reduction with test item: No colour change was observed after three hours of incubation during the check for possible direct MTT reduction by the test item. The test item did not interact with MTT, therefore additional controls and data corrections 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, light yellow 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. Two additional test item-treated tissues were used for the non-specific OD evaluation. The Non Specific Colourliving % (NSCliving %) was calculated as 1.5 %.

Validity of the Test
- The mean OD value of the two negative control tissues was: 2.161
- The positive control result showed 4.0 % viability at 30 minutes exposure.
- The difference of viability between the two relating tissue replicates ranged from 0.0 % to 1.3 %.
- The mean OD value of the blank sample isopropanol was 0.0384
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

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 (%)	Δ%
Negative Control: Sterile deionized water	1 2	2.169 2.152	100.4 99.6	0.8
	mean	2.161	100.0	-
Positive Control: Methyl Acetate	1 2	0.078 0.093	3.6 4.3	0.7
	mean	0.086	4.0	-

 

OD values and viability percentages of the test item:

Test item	Optical Density (OD)		Viability (%)	CFV NSCliving %	Δ%
Triethylene glycol dimethanesulfonate	1 2	0.179 0.206	8.3 9.5	6.8 8.1	1.3
	mean	0.192	8.9	7.4	-

 

OD values and NSCliving% of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour % (NSCliving %)	Δ%
Triethylene glycol dimethanesulfonate	1 2	0.032 0.031	1.5	0.0
	mean	0.032		-

Remark:
Δ%: The difference of viability between the two relating tissues
CFV NSCliving: Corrected Final Viability
Mean blank OD value was 0.0384

Interpretation of results:

other: Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1)

Conclusions:

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) indicate that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2.

Executive summary:

The purpose of this study was to determine the acute ocular irritation potential of the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) on three-dimensional RhCE tissue in the EpiOcular™ model in vitro.

Before the treatment the tissues were pre-wetted with 20 μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with the test item and incubated for 30 minutes (± 2 min) at standard culture conditions (37 ± 2 °C in an incubator with 5 ± 1 % CO2, ≥95 % humidified atmosphere).

Exposure of the test item was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 12 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, the test item treated tissues were incubated for 120 ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37 ± 2 °C in an incubator with 5 ± 1 % CO2 protected from light, ≥95 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol 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 (Optical Density) evaluation (NSCliving).

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 30 minutes (± 2 min).

The test item Triethylene glycol dimethanesulfonate showed significantly reduced cell viability in comparison to the negative control (corrected final viability: 7.4 %). All obtained test item viability results were below 60 % when compared to the viability values obtained from the negative control.

Positive control viability results (mean tissue viability: 4.0 %) were below 50 % when compared to the viability values obtained from the negative control. Furthermore, the mean optical density (OD) value of the two negative control tissues (mean OD value: 2.161) were between 0.8 and 2.8. So, the positive and negative controls showed the expected values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) indicate that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

From 25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

Amendment Commission Regulation (EC) No 2017/735, dated 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni utca 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Only the eyes of healthy animals considered suitable for entry into the human food chain were used. Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (20.3 ºC to 21.2 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were visually inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. Cornea integrity was checked by applying one small drop of fluorescein 2 % (w/v) solution onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 µL

POSITIVE AND NEGATIVE CONTROL
- Positive control: 30 µL
- Negative control: 30 µL

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Test item and positive control samples: Three replicates
Negative control samples: One replicate

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. Cornea integrity was checked by applying one small drop of fluorescein 2 % (w/v) solution onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

- Preparation of eyes
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

- Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again, avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ± 5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0 % to 2 %) were observed in the eyes, finding considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. None of the eyes were discarded as no eye was considered unsuitable after the baseline assessment.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position and 30 µL of test item was applied from micropipette onto the center of the cornea, taking care not to damage or touch the cornea with the application equipment. This procedure was repeated with the remaining two test item treated eyes.
The three positive control eyes were treated with (30 µL/eye) benzalkonium chloride solution (5 %) and one negative control eye was treated with 30 µL isotonic saline [NaCl (9 g/L saline)] according to the above procedure.

OBSERVATION PERIOD
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
The cornea thickness was measured and cornea opacity and possible morphological effects were examined at all time points. Fluorescein retention was determined at baseline (t = 0) and 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL isotonic saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
The cornea surface of negative and positive control treated corneas was also rinsed thoroughly after an exposure period of 10 seconds with 20 mL isotonic saline solution at ambient temperature, while taking care not to damage the corneas.

DECISION CRITERIA:
The conclusion on eye irritancy was based on the criteria given in OECD 438 guideline on quantitative assessments.

Irritation parameter:

percent corneal swelling

Remarks:

mean maximum corneal swelling up to 75 min

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Maximum corneal swelling at up to 75 min: 0 %

Positive controls validity:

valid

Remarks:

Mean maximum corneal swelling at up to 75 min: 25 %

Remarks on result:

other: ICE Class I

Irritation parameter:

percent corneal swelling

Remarks:

mean maximum corneal swelling up to 240 min

Run / experiment:

mean

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Maximum corneal swelling at up to 240 min: 0 %

Positive controls validity:

valid

Remarks:

Mean maximum corneal swelling at up to 240 min: 42%

Remarks on result:

other: ICE Class I

Irritation parameter:

cornea opacity score

Remarks:

mean maximum corneal opacity

Run / experiment:

mean

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Maximum corneal opacity: 0.0

Positive controls validity:

valid

Remarks:

Mean maximum corneal opacity: 4.0

Remarks on result:

other: ICE Class I

Irritation parameter:

fluorescein retention score

Remarks:

Mean fluorescein retention

Run / experiment:

mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Fluorescein retention: 0.0

Positive controls validity:

valid

Remarks:

Mean fluorescein retention: 3.0

Remarks on result:

other: ICE Class I

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.
- Acceptance criteria met for positive control: The positive control Benzalkonium chloride solution 5 % (w/w) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Interpretation of results:

other: No Category

Conclusions:

In this ICET, the overall ICE classes were thrice I. According to the guideline OECD 438, Triethylene glycol dimethanesulfonate (CAS 80322-82-3) is categorized as “No Category”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 μL/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test item exposure) were determined at each of the above time points.

The test item Triethylene glycol dimethanesulfonate, the positive control [Benzalkonium chloride solution (5 %)], and the negative control (NaCl, 9 g/L saline) were applied in a volume of 30 μL/eye, in such a way to evenly cover the whole cornea surface of each tested eye. Three test item treated eyes, three positive control eyes and one negative control eye were used in this study.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

 

In this ICET, the overall ICE classes of the test item were thrice I (based on the corneal swelling of 2 % within 240 minutes, based on the corneal opacity score of 0.2 and based on the fluorescein retention of 0.5).

The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. Furthermore, the three endpoints of the positive and the negative control were in the historical control range. So, the positive and negative controls showed the expected results. The experiment was considered to be valid.

In this ICET, the overall ICE classes were thrice I. According to the guideline OECD 438, Triethylene glycol dimethanesulfonate (CAS 80322-82-3) is categorized as “No Category”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation:

A valid EpiDerm^™ test of the test item Triethylene glycol dimethanesulfonate (CAS-No. 80322-82-3) has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, 18 June 2019. EpiDerm discs were exposed in duplicates to the test item for 60 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. 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. 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. The results indicate that the test item is not corrosive and according to the UN GHS classification system, Triethylene glycol dimethanesulfonate can be classified as “Non-corrosive”.

 

A valid EpiDerm^™ Model test of Triethylene glycol dimethanesulfonate (CAS 80322-82-3) 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, 14 June 2021. EpiDerm discs were exposed in triplicate to the test item for 60 min (25 min at room temperature followed by 35 minutes at 37±1 °C, 5±1 % CO2, 90±10 % humidified atmosphere). The test item has an intrinsic colour (dark yellow), therefore two additional test item treated tissues were used for the non-specific optical density (OD) evaluation (NSCliving) in all experiments. The EpiDerm discs treated with the test item did not show significantly reduced cell viability in comparison to the negative control in the first (mean relative viability: 59.2 %) and the additional experiment (mean relative viability: 79.9 %). All obtained test item viability results were above 50 %, when compared to the viability values obtained from the negative control in each run. Therefore, the test item was considered to be non-irritant to skin and is not classified (UN GHS / CLP No Category) according to the guideline OECD 439.

 

Eye irritation:

A valid study was performed under GLP to determine the acute ocular irritation potential of the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) on three-dimensional RhCE tissue (EpiOcular^™ model) in vitro according to OECD guideline No. 492, 18 June 2019. The test item has an intrinsic colour (dark yellow), therefore two additional test item treated tissues were used for the non-specific OD (Optical Density) evaluation (NSCliving). Disks of EpiOcular™ (two units) were treated with 50µL test item for the plates with the treated tissue units were incubated for an exposure period of 30 minutes at (±2 min) standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere).  
The test item Triethylene glycol dimethanesulfonate showed significantly reduced cell viability in comparison to the negative control (corrected final viability: 7.4 %). All obtained test item viability results were below 60 % when compared to the viability values obtained from the negative control. The results obtained from this in vitro eye irritation test, using the EpiOcular^™ model, with the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) indicate that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2.

 

A valid Isolated Chicken Eye Test (ICET) was performed according to the OECD Test guideline No. 438, 25 June 2018 to evaluate the potential ocular corrosivity and irritancy of the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose of 30 µL to three corneas and rinsed off after exposure period of 10 seconds. Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. In this ICET, the overall ICE classes of the test item were thrice I (based on the corneal swelling of 2 % within 240 minutes, based on the corneal opacity score of 0.2 and based on the fluorescein retention of 0.5). Thus, the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) is categorized as “No Category” according to the guideline OECD 438.

Justification for classification or non-classification

For Skin Irritation:

According to the results obtained with the EpiDerm™ tests for skin corrosion and skin irritation (OECD 431 and OECD 439) the test item is considered to be not corrosive and non-irritant to skin and is therefore not classified (UN GHS / CLP No Category) according to the CLP Regulation (EC) No 1272/2008.

 

For Eye Irritation:

The Epiocular™ test (OECD 492) indicated that the test item is considered as Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1) under the applied testing conditions. However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. The Isolated Chicken Eye (ICE) test performed showed that the test item has to be categorized as “no Category”. Therefore, it is concluded in a WoE approach that the test item has to be classified as irritating to the Eye (UN GHS Category 2) according to the CLP Regulation (EC) No 1272/2008.