Potassium thiosulphate

Endpoint summary

Description of key information

skin irritation/ corrosion: not irritating, No Category (OECD 439; GLP)

eye irritation/ corrosion: not irritating, No Category (OECD 491; GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-04-20 to 2021-05-21

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:

2020-06-26

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-02-10

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2019-10-23

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (Standard Assay Kit and MTT-100 kit; MatTek)
- Tissue lot number: 34158

TEST FOR MTT INTERFERENCE
- 1 ml of a MTT solution (1 mg/mL) including 50±2 mg of the test item was incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2).
- Untreated MTT/DMEM solution (1 mg/mL) was used as negative control.
- After incubation the change of colour was determined by the unaided eye.

TEST FOR COLOUR INTERFERENCE
- 50 ± 2 mg of the test item was added to 1 mL of deionised water and mixed. 1 mL of deionised water was used as control (blank). Both were incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2).
- 50 ± 2 mg of the test item was added to 2 mL of isopropanol and mixed. A control (2 mL of isopropanol, blank) was run concurrently. Both were incubated for 60 min at room temperature.
- After incubation the change of colour was determined by the unaided eye.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE AND COLOUR INTERFERENCE
Since the test item interfered with MTT and reduced it to formazan by itself two additional controls in duplicates run with the main experiment – the freeze-killed tissue controls (killed controls = KC; supplied by MatTek):
- Deionised water treated freeze-killed tissues (NC_KC)
- Test item treated freeze-killed tissues (TI_KC)
At the end, the following data correction procedure for Viability plus killed controls were performed:
Corrected viability (%) = TI viability – (TI_KC viability – NC_KC viability)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min, followed by incubation at room temperature until the 60 min treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1.5 °C

DETAILS OF THE TEST PROCEDURE USED
- Pre-warming:
The inserts with the EpiDerm™ tissues were transferred into 6-well-plates containing 0.9 ml assay medium and incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2). Afterwards, the medium was changed and a further pre-incubation for 21 h and 20 min (37 ± 1.5°C and 5 ± 0.5% CO2).
- Treatment:
The EpiDerm™ tissues were wetted with 25 µL DPBS prior to application. 25 ± 2 mg (39.7 mg/cm2) of the test item and 30 μL of the positive and negative control, respectively, were applied onto the surface of the tissue. Within the exposure time of 60 min the 6-well plates were placed in the incubator for 35 min (37 ± 1.5°C, 5 ± 0.5% CO2) and the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment. At the end of the treatment interval the tissues were gently rinsed with PBS (in-house) several times in order to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
-Post-incubation:
Afterwards, the tissues were incubated in 0.9 mL of fresh assay medium for 23 h and 36 min. After this incubation period the medium was changed, and the tissues were incubated for another 18.5 h. The complete post-incubation time was 42 h and 6 min (37 ± 1.5°C, 5 ± 0.5% CO2).

MTT ASSAY
- For the MTT-assay 24-well plates were prepared with 300 µl MTT (1 mg/mL) solution per well. After post-incubation all tissues were transferred to the prepared MTT-plates. After a 3 h ± 5 min incubation period (37 ± 1.5°C, 5 ± 0.5% CO2) the tissues were rinsed with PBS and carefully dried with blotting paper.
- The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted within 2 h and 42 min while shaking at room temperature.
- Per each tissue, 3 x 200 µL aliquots of the formazan solution were transferred into a 96-well, flat bottom, microtiter plate for OD measurement. 200 µL of isopropanol were added to the wells designated as blanks for 96-well plate.
- Measurement: The optical density (OD570nm) was determined spectrophotometrically in duplicates by a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
The main experiment was performed once.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Overall remarks, attachments" below.

PREDICTION MODEL / DECISION CRITERIA
According to OECD 439:
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponded to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [(mean ODtest item / positive control) / ODmean of negative control] * 100
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control. The test item is considered to be irritant to skin or serious eye damaging in accordance with Regulation EC No. 1272/2008 (UN GHS “Category 1 or 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. In this case further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification. The test substance may be considered as non-irritant to skin in accordance with Regulation EC No. 1272/2008 (UN GHS “No Category”) if the tissue viability after exposure and post-treatment incubation is more than 50%.

TEST ACCEPTANCE CRITERIA
According to OECD 439:
- the mean OD570 of the tissue replicates treated with the negative control should be ≥ 0.8 and ≤ 2.8
- the viability of the tissue replicates treated with the positive control should be ≤ 20%
- the standard deviation calculated from 3 identically treated replicates should be ≤ 18

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the skin irritation potential of the proficiency substances listed in Table 1 of OECD TG 439. The respective proficiency data are attached in the field "Overall remarks, attachments" below.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: The test item was tested neat. Three tissue replicates were wetted with 25 µL of DPBS prior to application and then 25 ± 2 mg (39.7 mg/cm²) of the test item were applied uniformly to the epidermis surface.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h and 6 min

Number of replicates:

test item, positive and negative control: in triplicate
additional freeze killed controls: in duplicate

Irritation / corrosion parameter:

% tissue viability

Value:

100.57

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

TEST FOR MTT INTERFERENCE
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed colour change. Therefore, an additional test with freeze-killed tissues was necessary for the quantitative correction of the test item viability.

TEST FOR COLOUR INTERFERENCE
The mean OD of the test item in deionised water or isopropanol was < 0.08 and therefore, an additional test with viable tissues without MTT addition was not necessary in the main experiment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of EpiDermTM for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the ten proficiency chemicals listed in Table 1 of OECD TG 439. The respective Laboratory Technical Proficiency Data are attached in the field "Overall remarks, attachments" below.

ACCEPTANCE OF RESULTS:
- the mean OD570 of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 (1.968) in accordance with OECD TG 439.
- the viability of the tissue replicates treated with the positive control is ≤ 20% (3.64).
- the standard deviation calculated from 3 identically treated replicates is ≤ 18 (0.3 – 4.1).
- Concurrent negative controls and positive controls demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) were within the defined historical acceptance ranges. The results of the positive and negative controls demonstrate reproducibility over time.

Please also refer to the field "Overall remarks, attachments" below.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in the described EpiDerm study and under the experimental conditions reported, the test item potassium thiosulfate is not irritating to skin and does not require classification and labelling for skin irritation or skin corrosion according to UN GHS and EU CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-07-29 to 2021-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 491 (Short Time Exposure In Vitro 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:

2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2022-11-01

Details on test animals or tissues and environmental conditions:

CELL CULTURE
- Cell Line SIRC: The rabbit corneal cell line SIRC (Statens Seruminstitut Rabbit Cornea) was used for performing the STE test method. SIRCs are growing as confluent monolayers.
- Large stocks of the SIRC cell line (supplied by ATCC) were stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells.
- Thawed stock cultures were propagated at 37 ± 1.5 °C in plastic flasks containing a culture medium comprising Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 units/mL penicillin and 100 µg/mL streptomycin.
- The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Cells were propagated 2 to 3 passages in a culture flask before being employed for testing and did not exceed 25 passages from thawing.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

other:

Amount / concentration applied:

TEST MATERIAL
On the day of the experiments right before application, the test item was dissolved in physiological saline (0.9% NaCl in deionised water) to reach a final concentration of 5% (w/w). Next, this solution was diluted by serial 10 fold dilution with the respective solvent to reach final concentrations of 0.5% (w/w) and 0.05% (w/w). The test item was prepared freshly prior to each repetition.

VEHICLE
physiological saline (0.9% NaCl in deionised water)

Duration of treatment / exposure:

5 minutes at room temperature

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

triplicate

Details on study design:

CELL LINE
Please refer to the field „Details on test animals or tissues and environmental conditions“

SEEDING OF THE CULTURES
Exponentially growing stock cultures more than 50% confluent were rinsed with PBS and treated with Trypsin at 37 ± 1.5 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete medium and a single cell suspension was prepared.
Individual wells of a 96-well tissue-culture microtiter plate were inoculated with 0.2 mL complete medium containing approximately 3 x 10E4 cells/mL (6000 cells per well) in case that the cells were seeded four days prior to the treatment and 1.5 x 10E4 cells/mL (3000 cells per well) in case that the cells were seeded 5 days prior to the treatment. The seeding day is day 0 and the day of treatment are included in the calculation of the days for the cell cultivation: e.g. seeding on Friday of 6000 cells/well and treatment on Tuesday (four days) or seeding on Friday of 3000 cells/well and treatment on Wednesday (five days). The plates will be incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Cells should have reached a confluence of more than 80% at the time of testing.

NUMBER OF REPETITIONS AND REPLICATES
The test item was tested in six independent repetitions with different cell cultures and/or on different days. All dose groups were tested in triplicates in each repetition. The first three independent repetitions were not valid and had to be repeated. Only the three valid repetitions (4,5 and 6) were reported.

TREATMENT
For the treatment the complete medium was removed, and the cells were re-fed with 200 µL treatment solution containing medium, solvent and positive control as well as the two different concentrations of the test item (5% and 0.05%) and the complete medium blank, respectively. In addition, in one dose group empty wells without any cells were treated with the test item and treated exactly the same as the other groups.

CELL VIABILITY MEASUREMENT
After exposure, cells were washed twice with 0.2 mL of calcium- and magnesium-free PBS and 0.2 mL MTT solution (0.5 mg MTT/mL of MEM) was added. After a two-hour ± 15 minutes reaction time at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere the MTT solution was decanted. MTT formazan was extracted by adding 0.2 mL of 0.04 N hydrochloric acid – isopropanol for at least 60 minutes (not longer than 120 minutes) in the dark at room temperature, and the absorbance of MTT formazan solution was measured with a microplate reader (Versamax® Molecular Devices) at 570 nm (without a reference). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DECISION CRITERIA
The cell viability cut-off values for identifying test items inducing serious eye damage (UN GHS category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS no category) correspond to Table 2 of the OECD TG 491.

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the eye irritation potential of the proficiency substances listed in Table 1 of OECD TG 491. The respective proficiency data are attached in the field "Overall remarks, attachments" below.

REFERENCE TO HISTORICAL POSITIVE CONTROL MEAN AND STANDARD DEVIATION (SD)
Historical data are available to derive comparable run acceptance criteria are attached in the field "Overall remarks, attachments" below.

TEST ACCEPTANCE CRITERIA
According to OECD TG 491:
- Optical density of the medium control should be 0.3 or higher after subtraction of blank optical density.
- Viability of the solvent control should be 80% or higher relative to the medium control.
- The cell viability of the positive control should be within the upper and lower acceptance boundaries established by the method developer (21.1% and 62.3%) in compliance with the OECD TG 491.
- Standard deviation of the final cell viability derived from three independent repetitions should be less than 15% for both 5% and 0.05% concentrations of the test chemical.

Irritation parameter:

other: Mean Cell Viability [%]

Run / experiment:

Test Item 0.05%

Value:

88.47

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Medium control: valid

Irritation parameter:

other: Mean Cell Viability [%]

Run / experiment:

Test Item 5%

Value:

95.16

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Medium control: valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the eye irritation potential of the chemicals listed in Table 1 of OECD TG 491. The respective proficiency data are attached in the field "Overall remarks, attachments" below.

TEST ACCEPTANCE CRITERIA
- Optical density of the medium control after subtraction of blank optical density was higher than 0.3 (0.355 - 0.759).
- Viability of the solvent control relative to the medium control was higher than 80% (93.3 - 98.5).
- Standard deviation of the final cell viability derived from three independent repetitions was less than 15% for both 5% and 0.05% test item concentrations (7.8 and 10.7).
The above acceptance criteria were met.
- The cell viability of the positive control (16.49%) was not within the upper and lower acceptance boundaries established by the method developer (21.1% and 62.3%) in compliance with the OECD TG 491. Regarding this acceptance criteria the positive control was too low. From a scientific point of view this does not affect the validity of the study, especially since a negative result was obtained for the test item. A repetition should provide higher OD values for the positive control that would finally lead to an increase of the OD level of the test item treated group, too. Higher OD values for the test item still would result in “No category”. Therefore, it can be assumed that a too low positive control stands for a sensitive cell passage and therefore the result for the test item is convincing and reliable.
Please also refer to the field "Overall remarks, attachments" below.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in the described STE study under the experimental conditions reported, the test item potassium thiosulfate is not eye irritating and does not require classification and labelling for eye irritation or serious eye damage according to UN GHS and EU CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The substance was not observed to be a skin irritant in a reliable in vitro study according to OECD 439.

Eye irritation:

The substance was not observed to be an eye irritant in a reliable in vitro study according to OECD 491.

Justification for classification or non-classification

Skin irritation:

The substance does not possess a skin irritation potential based on an in vitro OECD 439 study and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

The substance does not possess an eye irritation potential based on an in vitro OECD 491 study and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.