(R*,R*)-1,4-dimercaptobutane-2,3-diol

Endpoint summary

Description of key information

• Episkin test for skin irritation (OECD 439): 6% cell viability: Irritant and/or corrosive.
• Episkin test for skin corrosion (OECD 431): Cell viabilities of 77, 68 and 57% after 3 min, 1h and 4h: Not corrosive. 
• ICE test (OECD 438): overall ICE classes were once II and twice III: No prediction can be made.
• Epiocular^TM(OECD 492): 4 % cell viability, Irritant and/or corrosive.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4-5 May 2020

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:

14 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: Human Skin Model Test"

Version / remarks:

Official Journal of the European Union No.
L142, dated May 31st, 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECVAM Database Service on Alternative Methods to Animal Experimentation, INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test”

Version / remarks:

Updated December 2011 / February 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch-No.: 44606300 / 50774
Storage: refrigerator, 2-8°C, under Nitrogen

Test system:

human skin model

Remarks:

EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN Laboratories Lyon, France

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Remarks:

100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: EPISKIN Laboratories, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 20-EKIN-010
- Expiry date: 09 March 2020
- Date of initiation of testing: 4 May 2020

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated over night at 37°C in an incubator with 5 % CO2, >= 95% humidified atmosphere.

APPLICATION / EXPOSURE
- Test Item: An amount of 20 mg test item was applied evenly to the epidermal surface of the two test skin units/exposure times respectively. Subsequently, 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.
- Positive and negative control: A volume of 50 µL positive control (glacial acetic acid) or negative control (NaCl 9 g/L) was 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 if necessary.
- Additional controls for MTT direct interacting chemicals: In addition to the normal procedure, 2 killed test item treated tissues and 2 killed negative control treated tissues were used for the MTT evaluation in one run in each exposure time (4h, 1h, 3min).
- Exposure: The plates with the treated epidermis units were incubated for the exposure time of 4 h ours at room temperature (22.7-23.1°C). Additional controls for MTT evaluation were also tested with each exposure time (4h, 1h, 3min).

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Duplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 ml PBS 1x Solution, twice (the one extra rinsing was made based on the above way). The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT TEST:
After the exposure of test item was terminated by rinsing with PBS, the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION:
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate). The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all epidermis material with the acidified isopropanol then incubated overnight at room temperature protected from light for formazan extraction. At the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIABILITY MEASUREMENTS:
Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (Linearity range: 0.2720 – 3.4218) using acidified isopropanol solution as the blank (6×200 µL).
Linear OD range of the spectrometer: not reported

NUMBER OF INDEPENDENT TEST SEQUENCES/ EXPERIMENTS TO DERIVE FINAL PREDICTION:
1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin (SubCat.1A) if mean tissue viability is < 35 % after 3 min exposure
- The test substance is considered to be corrosive to skin (subCat.1B or 1C) if Mean tissue viability is = 35 % after 3 min exposure and < 35 % after 1 hour exposure OR Mean tissue viability is = 35 % after 1 hour exposure and < 35 % after 4 hours exposure
- The test substance is considered to be non-corrosive to skin if mean tissue viability is = 35 % after 4 hours exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg

VEHICLE
- no, but 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis.

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): NaCl 9 g/L

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

4h, 1h and 3min for test item and negative control
4h for the positive control

Duration of post-treatment incubation (if applicable):

no post-treatment incubation

Number of replicates:

Two replicates were used for the test item and control(s) respectively.

Irritation / corrosion parameter:

% tissue viability

Remarks:

4h exposure

Run / experiment:

1

Value:

57

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

difference of viability between the two tissue replicates: 23.5

Positive controls validity:

valid

Remarks:

2% viability, difference of viability between the two tissue replicates: 0.9%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

1h exposure time

Run / experiment:

1

Value:

68

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% viability, difference of viability 22%

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min exposure

Run / experiment:

1

Value:

77

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% viability, difference of viability 7.3%

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 31.61 %, 34.76 % and 22.36 % at the 4h, 1h and 3 min exposure respectively. As the NSMTT were below 50 % in each case the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. Furthermore, the test item was completely removed from the epidermal surface at rinsing per iod. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
-Acceptance criteria met for negative control (4h exposure): yes, OD value 0.791, difference of viability between the two tissue replicates: 23.4% (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be < or = 30)
- Acceptance criteria met for positive control (4h exposure): yes, 0.016 % viability, difference of viability between the two tissue replicates: 0.9% (The acceptable mean percentage viability range for positive controls is 0-20% and the standard deviation value (SD) of the % viability should be < or = 30.)
- Acceptance criteria met for variability between replicate measurements (4h exposure): yes, difference of viability between the two tissue replicates test item: 18 % (should be < or = 30)

OD values and cell viability percentages of the positive and negative control

Controls	Optical Density (OD)		Viability (%)	Delta%
Negative control NaCl (9g/L saline) 4h exposure	1 2 mean SD CV	0.791 1.000 0.895 0.148 16.518	88 112 100 16.518 16.518	23.4
Negative control NaCl (9g/L saline) 1h exposure	1 2 mean SD CV	1.005 0.806 0.905 0.141 15.566	111 89 100 15.566 15.566	22.0
Negative control NaCl (9g/L saline) 3 min exposure	1 2 mean SD CV	0.918 0.988 0.953 0.049 5.157	96 104 100 5.157   5.157	7.3
Positive Control: Glacial acetic acid 4 h exposure	1 2 mean SD CV	0.012 0.020 0.016 0.005 34.754	1 2 2 0.608 34.754	0.9

Remark: delta%: The difference of viability between the two relating tissues.

Mean blank OD values were 0.0386 each plate

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

OD Values and viability percentages of the test item

Test Item	Optical Density (OD)		TODTT	Viability (%)	Delta%	Relative viability (%)
1,4-Dithiothreitol 4h exposure	1 2 mean SD CV	0.916 0.755 0.835 0.114 13.654	0.552 0.471 0.512 0.057 11.137	102 84 93 12.742 13.654	18.0	62 53 57 6.371 11.137
1,4-Dithiothreitol 1h exposure	1 2 mean SD CV	0.905 0.944 0.924 0.027 2.956	0.610 0.629 0.619 0.014 2.206	100 104 102 3.019 2.956	4.3	67 69 68 1.509 2.206
1,4-Dithiothreitol 3 min exposure	1 2 mean SD CV	0.845 0.983 0.914 0.098 10.720	0.701 0.770 0.736 0.049 6.660	89 103 96 10.284 10.720	14.5	74 81 77 5.142 6.660

Delta%: The difference of viability between the two relating tissues

TOD_TT: true MTT metabolic conversion

Mean blank OD values were 0.0386 each plate

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

OD values of additional controls for MTT-interacting test item

Controls	Optical Density (OD)		NSMTT %
Negativ control killed tissues: NaCl (9 g/L saline) 4h exposure	1 2 mean SD CV	0.129 0.125 0.127 0.003 2.532	31.61
Test Item treated killed tissues: 1,4-Dithiothreitol 4h exposure	1 2 mean SD CV	0.441 0.379 0.410 0.044 10.665
Negativ control killed tissues: NaCl (9 g/L saline) 1h exposure	1 2 mean SD CV	0.125 0.129 0.127 0.003 2.113	34.76
Test Item treated killed tissues: 1,4-Dithiothreitol 1h exposure	1 2 mean SD CV	0.456 0.427 0.442 0.021 4.681
Negativ control killed tissues: NaCl (9 g/L saline) 3 min exposure	1 2 mean SD CV	0.166 0.159 0.162 0.005 2.829	22.36
Test Item treated killed tissues: 1,4-Dithiothreitol 3 min exposure	1 2 mean SD CV	0.449 0.301 0.376 0.103 27.547

NSMTT %: non-specific MTT reduction %

Mean blank OD values were 0.0386 each plate

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

Interpretation of results:

other: The test substance can be classified as Non-corrosive. However, skin irritation potential cannot be excluded.

Remarks:

The study is used for classification in a weight of evidence approach.

Conclusions:

In this in vitro skin corrosion test in EPISKIN model (OECD 431) with 1,4-Dithiothreitol (CAS-No. 3483-12-3) the results indicate that the test item is not corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time. In conclusion, the test item 1,4-Dithiothreitol can be classified as Non-corrosive. However, skin irritation potential cannot be excluded. Further studies are needed for classification.

Executive summary:

Disks of EPISKIN (two units / chemical / incubation time) were treated with the test item and incubated for 4 hours (±10 min), 1 hour (±5 min) and 3 min at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a = 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively.

The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement.

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 4 hours of exposure is above or equal 35 % of the negative control.

Each test item treated tissue viabilities were above 35 % of the mean negative control value after 4 hours, 1 hour and 3 minutes exposure. The average test item treated tissue relative viabilities were 57 % at 4 hours, 68 % at 1 hour and 77 % at 3 minutes of exposure.

Positive and negative controls showed the expected 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 EPISKIN model (OECD 431) with 1,4-Dithiothreitol (CAS-No. 3483-12-3) the results indicate that the test item is not corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time. In conclusion, the test item 1,4-Dithiothreitol can be classified as Non-corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 - 22 May 2020

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:

18 June 2019

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:

10 February 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No: 44606300 / 50774
Storage: refrigerator, 2-8 °C, under Nitrogen

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, Slovakia
- Tissue batch number(s): 30866
- Expiry date: May 22, 2020
- Date of initiation of testing: May 20, 2020

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells
were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the
media below them, in contact with the epidermis into each prepared well and then incubated over
night at 37°C in an incubator with 5 % CO2, >= 95% humidified atmosphere.

EXPOSURE
- Test Item: Before application, the test item was ground to obtain a fine powder. First, 25 µL of sterile DPBS was applied to the epidermal surface in order to improve further contact between powder and epidermis. Subsequently, 25 mg of the test item was applied evenly to the epidermal surface. The insert was gently shaken from side to side to ensure that the tissue was completely covered by the test item.
- 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. Application of the nylon mesh was not necessary..
-Additional controls for MTT direct interacting chemicals: In addition to the normal procedure, 2 killed test item treated tissues and 2 killed negative control treated tissues were used for the MTT evaluation in one run.
- Exposure times: The total exposure time was 60 minutes, which included two 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.

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS:
Three replicates were used for the test item and positive and negative controls, respectively.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure time (60±1 min) the EpiDermTM units were 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).
- Observable damage in the tissue due to washing: none

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 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled
wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an i
ncubator with 5 % CO2 protected from light, =95% humidified atmosphere.

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 repeated twice. After the last aspiration the dryness of the tissue surface was checked 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 was 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25mg
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 and controls, respectively.

Irritation / corrosion parameter:

% tissue viability

Value:

12

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean viability value of three tissues

Other effects / acceptance of results:

OTHER EFFECTS:
- Possible direct MTT reduction with test item:
During the check-method for possible direct MTT reduction (see section 5.5.1), colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary.
The non-specific MTT reduction (NSMTT) was determined to be 5.9 %. As the NSMTT were below 30 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colouring potential of test item
The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered not to be able to significantly stain the tissues and lead to a false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD value 2.323 and standard deviation value (SD) 11.13 for the100 % viability (The mean OD value of the three negative control tissues should be between 0.8 and 2.8 and the standard deviation value (SD) of the % viability should be = 18.)
- Acceptance criteria met for positive control: Mean OD value 0.039 and standard deviation value (SD) 0.07 for the 2 % viability (The acceptable percentage viability for positive control (mean of three tissues) is < 20 % and the standard deviation value (SD) of the % viability should be = 18)
- For the test item, the standard deviation value (SD) of the % viability should be = 18.

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:

Substance	Optical Density (OD)		Viability (%)
Negative Control 1x DPBS	1 2 3 mean standard deviation (SD)	2.062 2.328 2.579 2.323	89 100 111 100 11.13
positive control SDS (5% aq.)	1 2 3 mean standard deviation (SD)	0.037 0.039 0.040 0.039	2 2 2 2 0.07

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

Substance	Optical Density (OD)		TODTT	Viability (%)	relative viability (%)
Test item 1,4 -Dithiothreitol	1 2 3 mean standard deviation (SD)	0.301 0.250 0.279 0.277	0.163 0.112 0.141 0.139	13 11 12 12 1.11	7 5 6 6 1.11

OD values of additional controls for MTT-interacting test item:

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x DPBS	1 2 mean standard deviation (SD)	0.045 0.041 0.043 0.14
Test item treated killed tissues: 1,4-Dithiothreitol	1 2 mean standard deviation (SD)	0.184 0.178 0.181 0.20

Interpretation of results:

other: Test item is irritant (UN GHS Category 2) and/or corrosive (UN GHS Category 1).

Remarks:

The study is used for classification in a weight of evidence approach.

Conclusions:

The results obtained from this in vitro skin irritation test, using the EpiDermTM model, with the test item 1,4-Dithiothreitol (CAS 3483-12-3) indicated that the test item is Irritant (UN GHS / CLP Category 2) and/or Corrosive (UN GHS /CLP Category 1). However, this test method (OECD 439) cannot resolve between UN GHS /CLP Categories 1 and 2.

Executive summary:

EpiDermTM Model test of 1,4-Dithiothreitol (CAS 3483-12-3) 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, 18 June 2019.

Disks of EpiDermTM (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 test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement.

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 EpiDermTM model, the test item 1,4-Dithiothreitol showed significantly reduced cell viability in comparison to the negative control (mean relative viability value: 6 %). All obtained test item viability results were far below 50 %, when compared to the viability values obtained from the negative control.

Positive and negative controls showed the expected optical density (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 EpiDermTM model, with the test item 1,4-Dithiothreitol (CAS 3483-12-3) indicated that the test item is Irritant (UN GHS / CLP Category 2) and/or Corrosive (UN GHS / CLP Category 1). However, this test method (OECD 439) cannot resolve between UN GHS / CLP Categories 1 and 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

3 April 2020

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:

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.: 44606300 / 50774
Storage: refrigerator, 2-8°C, under Nitrogen

Species:

chicken

Strain:

other: ROSS 308

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: 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 (19.3 ºC to 20.3 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 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)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03g

POSITIVE AND NEGATIVE CONTROL
-Positive control: 0.03g
-Negative control: 30uL

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:

DETAILS ON TEST SYSTEM
- Strain of chicken: ROSS 308
- Supplier: TARAVIS KFT. 9600 Sárvár, Rábasömjéni utca 129. Hungary
- Handling and Storage: 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 (19.3 ºC to 20.3 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 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).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, it is used as part of a tiered testing strategy for regulatory purposes. In this assay, test items that are positive can be classified as ocular corrosives or severe irritants (UN GHS Category 1) while clearly negative test items can be identified as not requiring classification and labelling (UN GHS No Category) without further testing.

DETAILS ON THE TEST PROCEDURE USED
- 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. One small drop of fluorescein solution 2 (w/v) % was applied 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 hand-held slit lamp or 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: 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.

- Acclimatization: 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.

-Baseline assessment:
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: After the zero reference measurements in each experiment, 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 over a container to catch waste, while the test item was applied onto the center of the cornea. The test item was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test item, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eye.
The three positive control eyes were treated in a similar way with 0.03 g Imidazole.
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 µL from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.

-Exposure: 10 seconds

-Rinsing: 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 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.

- Post-rinsing observation period: The gentle rinsing with 20 mL saline was performed in all test item and positive control treated eyes after the 30, 75, 120 and 180 minutes of observation.

- Observation and assessment of corneal effects: 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 and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t = 0) and 30 minutes after the post-treatment rinse.

- Histopathology: A histopathology of the corneas was not performed as not borderline results were obtained, for which histopathology could provide final clarification. Corneas are discarded 2 months after the final report.

Irritation parameter:

percent corneal swelling

Remarks:

mean maximum corneal swelling up to 75 min

Run / experiment:

1

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean maximum corneal swelling up to 75 min: 0 %

Positive controls validity:

valid

Remarks:

mean maximum corneal swelling up to 75 min: 33 %

Remarks on result:

other: ICE Class II

Irritation parameter:

percent corneal swelling

Remarks:

percent corneal swelling upt to 240 min

Run / experiment:

1

Value:

9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean maximum corneal swelling upt to 240 min: 0 %

Positive controls validity:

valid

Remarks:

mean maximum corneal swelling upt to 240 min: 40 %

Remarks on result:

other: ICE Class II

Irritation parameter:

cornea opacity score

Remarks:

Mean maximum corneal opacity

Run / experiment:

1

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean maximum corneal opacity: 0.0

Positive controls validity:

valid

Remarks:

Mean maximum corneal opacity: 4.0

Remarks on result:

other: ICE Class III

Irritation parameter:

fluorescein retention score

Remarks:

Mean fluorescein retention

Run / experiment:

1

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean fluorescein retention: 0.0

Positive controls validity:

valid

Remarks:

Mean fluorescein retention: 3.0

Remarks on result:

other: ICE Class III

Other effects / acceptance of results:

ACCEPTANCE OF TEST RESULTS
The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1.
The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.
Positive and negative control values were within the corresponding historical control data ranges.

Interpretation of results:

other: No prediction can be made

Remarks:

The study is used for classification in a weight of evidence approach.

Conclusions:

In this ICET, the overall ICE classes were once II and twice III. According to the guideline OECD 438, 1,4-Dithiothreitol (CAS 3483-12-3) is categorized as “No prediction can be made”.

Executive summary:

In this ICET, the overall ICE classes were once II (based on the corneal swelling of 9% within 240 minutes) and twice III (based on the corneal opacity score of 2.0 and fluorescein retention of 2.0).

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 controls 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 once II and twice III. According to the guideline OECD 438, 1,4-Dithiothreitol (CAS 3483-12-3) is categorized as “No prediction can be made”.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 - 18 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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:

10 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch-No.: 44606300 / 50774
Storage: refrigerator, 2-8°C, under Nitrogen

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 mg (approximately 83.3 mg/cm2 application).

Duration of treatment / exposure:

6 hours (± 15 min)

Duration of post- treatment incubation (in vitro):

25±2 minutes immersion incubation (Post-Soak)
18 hours ± 15 minutes (Post-treatment Incubation)

Number of animals or in vitro replicates:

two

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, 821 05 Bratislava, Slovakia
- Lot No.: 30663
- Expiry date: 18 June 2020
- Storage: The EpiOcular™ (OCL-200-EIT) units were stored at refrigerator (2-8°C) until the preparation of tissues for treatment is started. The assay media supplied with the kits were stored at 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 THE TEST PROCEDURE USED:
- 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 37±1 °C. 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 was 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 approximately 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 ground, 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues (approximately 83.3 mg/cm2 application). To avoid that test item is spilled into the medium under the tissue inserts, the tissue insert was removed from the medium, placed onto a sterile surface and dosed by pouring the solid test item onto the tissue surface so that the surface of the tissue was completely covered by the test item. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.
*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 MTT direct interacting chemicals: In addition to the normal procedure, 2 killed test item treated tissues and 2 killed negative control treated tissues were used for the MTT evaluation in one run.

- Exposure
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at 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 was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates 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 25±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 18 hours ± 15 minutes at standard culture conditions (Post-exposure Incubation).

- MTT Test After Post-incubation
After the post-incubation the EpiOcular™ units were transferred into the 24-well plate filled with MTT ready to use solution (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.

- Formazan Extraction
Inserts were 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 isopropanol in each well so that no isopropanol was flowing into the insert. The plate was sealed with parafilm and extracted.
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (~120 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive and additional 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 (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm using isopropanol as the blank (8×200 µL).

OTHER INFORMATION ON MATERIALS AND METHODS
- Check-method for possible direct MTT reduction by the test Item
Amount of 50 mg test item was added to 1mL of 1 mg/mL MTT solution in a tube and the mixture was incubated in the dark at 37±2 °C, 5±1 % CO2, = 95 % humidified atmosphere. The mixture was incubated for approximately 3 hours (±15 min) and then any colour change observed through an unaided eye assessment:
- Test item which does not interact with MTT: yellow
- Test item interacting with MTT: blue or purple
If the MTT solution turns blue or purple, the test item interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT, i.e. by using killed EpiOcular tissues.

- Additional controls for MTT interacting chemicals (MTT reducers)
In addition to the normal procedure, 2 killed test item treated tissues and 2 killed negative control treated tissues were used for the MTT evaluation in one run (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues (batch No. of killed EpiOcular™ tissues: 30659). The same treatment steps were followed for these tissues as for the living tissues.

- Freeze killed tissues for MTT reducers
* Placing untreated EpiOcular™ constructs (in a 24 well plate) in the -15 to -30 °C freezer overnight.
* Thawing to room temperature.
* Refreezing (-15 to -30 °C).
* Once frozen, the tissue may be stored indefinitely in the freezer.
* Before use, the killed tissues are de-frozen at room temperature.
* Further use of killed tissues is similar to living tissues.
* Stored between -15 to -30 °C

- Check-method to detect the colouring potential of test Item
Prior to treatment, the test item was evaluated for its intrinsic colour or its ability to become coloured in contact with water and isopropanol. The non-coloured (e.g. white, off-white, light yellow colorants) materials may be become colorants after contact with water or isopropanol.
Amount of 50 mg test item was added to 1 mL of water in a tube and the mixture was incubated in the dark at 37±2 °C, 5±1 % CO2, in a = 95 % humidified atmosphere for one hour and then colour was checked through an unaided eye assessment.
Amount of 50 mg test item was added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in tube. The tube was placed on an orbital plate shaker and shaken (~120 rpm) for approximately 2 hours at room temperature and then colour was checked through an unaided eye assessment.

- Assay acceptance criteria
- The mean OD value of the two negative control tissues should be between 0.8 and 2.8.
- The acceptable percentage viability for positive control (mean of two tissues) is:
6 hours exposure: below 50% of control viability (solids)
- The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: Mean Tissue Viability (% of negative control)

Value:

4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Possible direct MTT reduction with test item:
During the check-method for possible direct MTT reduction (see section 5.5.1), colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary.
The non-specific MTT reduction (NSMTT) was determined (0 %)*, the correction of viability percentage was not necessary.
*: The calculated mean NSMTT was -1.302 %. However, for the calculation of non-specific MTT reduction, small negative numbers are counted as zero, because the reason of the small negative number is a slight difference between the used killed epidermis (biological variability).

- Colouring potential of test item:
The test item showed no ability to become coloured in contact with water and isopropanol. The intrinsic colour of the test item is white and therefore considered not to be able to significantly stain the tissues and lead to a false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean OD value 1.094
- Acceptance criteria met for positive control: 9 % viability at 6 hours exposure
- Difference of viability between the two tissue replicates: 0.0 to 1.7 %

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

Negative Control: Sterile deionized water

Replicate	Optical Density (OD)	Viability (%)
1	1.094	100
2	1.095	100
mean	1.094	100

 

positive control: methyl acetate

Replicate	Optical Density (OD)	Viability (%)
1	0.089	8
2	0.108	10
mean	0.098	9

Test item: 1,4 -Dithiothreitol

Replicate	Optical Density (OD)	Viability (%)
1	0.045	4
2	0.045	4
mean	0.045	4

OD values of additional controls for MTT-interacting test item:

Negative control treated killed tissues: Sterile deionized water

Replicate	Optical Density (OD)
1	0.044
2	0.053
mean	0.045

Test item treated killed tissues: 1,4-Dithiothreitol

Replicate	Optical Density (OD)
1	0.033
2	0.036
mean	0.035

Interpretation of results:

other: The test item is irritant (UN GHS Category 2) and/or corrosive (UN GHS Category 1).

Remarks:

The study is used for classification in a weight of evidence approach.

Conclusions:

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item 1,4-Dithiothreitol (CAS 3483-12-3) indicate that the test item is Irritant (UN GHS / CLP Category 2) and/or Corrosive (UN GHS / CLP Category 1). However, this test method (OECD 492) cannot resolve between UN GHS / CLP Categories 1 and 2. Therefore further testing with other test methods is required to decide on its final classification.

Executive summary:

The purpose of this study was to determine the acute ocular irritation potential of the test item 1,4-Dithiothreitol (CAS 3483-12-3) on three-dimensional RhCE tissue in the EpiOcular™ model in vitro.

Before the treatment the tissues were pre-wetted with approximately 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 6 hours (± 15 min) at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, = 95 % humidified atmosphere).

Exposure of the test itemwas terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 25±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 18 hours ± 15 minutes at standard culture conditions (Post-exposure 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 is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement. However, the non-specific MTT reduction (NSMTT) was determined (0 %)*, the correction of viability percentage was not necessary.

*: The calculated mean NSMTT was -1.302 %. However, for the calculation of non-specific MTT reduction, small negative numbers are counted as zero, because the reason of the small negative number is a slight difference between the used killed epidermis (biological variability).

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 6 hours ± 15 minutes.

The test item 1,4-Dithiothreitol showed significantly reduced cell viability in comparison to the negative control (mean tissue viability: 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: 9 %) 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: 1.094) 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 1,4-Dithiothreitol (CAS 3483-12-3) indicate that the test item is Irritant (UN GHS / CLP Category 2) and/or Corrosive (UN GHS / CLP Category 1). However, this test method (OECD 492) cannot resolve between UN GHS / CLP Categories 1 and 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

A valid In Vitro Skin Irritation Test with 1,4-Dithiothreitol in the EpiDerm^TMModel was performed under GLP 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, 18 June 2019.

EpiDerm^TMdiscs were exposed in triplicate to 1,4-Dithriothreitol for 25 min at room temperature and 35 minutes at standard culture conditions (37±1 °C, 5±1 % CO2, 90±10 % humidified atmosphere). The EpiDerm^TMdiscs treated with the test item showed significantly reduced cell viability (mean relative viability value: 6 %). Therefore 1,4-Dithiothreitol is considered to be irritant and/or corrosive to skin. The study is used in a weight of evidence approach. The study is rated as key study and as Klimisch 1 "reliable without restriction".

 

A valid In Vitro Skin Corrosion Test with 1,4-Dithiothreitol in the EPISKIN Model was performed under GLP 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, 14 June 2019.

EPISKIN discs were exposed in duplicate to 1,4-Dithiothreitol for4 hours (±10 min), 1 hour (±5 min) and 3 min at room temperature. The EPISKIN discs treated with the test item did not showed reduced cell viability </= 35%, in comparison to the negative control (mean relative viability: 57 % at 4 hours, 68 % at 1 hour and 77 % at 3 minutes of exposure). In conclusion, the test item 1,4-Dithiothreitol can be classified as Non-corrosive. The study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

 

 

Eye irritation:

A valid study was performed under GLP to determine the acute ocular irritation potential of the test item 1,4-Dithriothreitol on three-dimensional RhCE tissue in the EpiOcular™ model in vitro, according to OECD guideline Nr. 492, 18 June 2019.

EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37±1°C) in an incubator with 5±1 % CO2, 90±10% humidified atmosphere). The Epiocular disks treated with 1,4-Dithriothreitol showed significantly reduced cell viability in comparison to the negative control (mean relative viability: 4 %). The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item 1,4-Dithriothreitol indicated that the test item is, thus, considered 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. Therefore, further testing was performed. The study is used in a weight of evidence approach. The study is rated as Klimisch 1 "reliable without restriction".

 

A valid assessment of ocular irritation of 1,4-Dithriothreitol with the in vitro Eye Irritation Test in isolated chicken eyes (ICET) was performed under GLP according the OECD guideline Nr 438 (25 June 2018).

1,4-Dithriothreitol was applied in a single dose (30 mg/eye) onto three corneas. Following exposure for 10 seconds, it was rinsed off. 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 were once II (based on the corneal swelling of 9% within 240 minutes) and twice III (based on the corneal opacity score of 2.0 and fluorescein retention of 2.0).   Thus, according to the guideline OECD 438, 1,4-Dithiothreitol (CAS 3483-12-3) is categorized as “No prediction can be made”. This study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

Justification for classification or non-classification

For Skin Irritation:

1,4-Dithriothreitol is considered to be irritant (UN GHS Category 2) and/or corrosive (UN GHS Category 1). However, the Episkin SM test (OECD 431) for corrosion potential categorised the test item 1,4-Dithriothreitol as Non-corrosive. Therefore1,4-Dithriothreitol has to be classified as Irritant to the skin (UN GHS Category 2) according to the CLP Regulation (EC) No 1272/2008.

For Eye Irritation:

The Epiocular test (OECD 492) indicated that 1,4-Dithriothreitol is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However this test can not distinguish between C1 and C2.

The ICE test (OECD 438) was performed. Using this test, no prediction could be made.

Therefore, as worst case, 1,4 -Dithriothreitol has to be classified as Corrosive (UN GHS Category 1) according to the CLP Regulation (EC) No 1272/2008.