Sodium thiosulphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-05 to 2021-02-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2020-06-26

Deviations:

no

Test guideline 2

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 material

Test material information

Constituent 1

Reference substance name:

Sodium thiosulphate

EC Number:

231-867-5

EC Name:

Sodium thiosulphate

Cas Number:

7772-98-7

Molecular formula:

H2O3S2.2Na

IUPAC Name:

sodium thiosulphate

Test material form:

solid: particulate/powder

Details on test material:

Appearance: white powder

In vitro test system

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: 34120 (for the quality certificate please also refer to the field "Overall remarks, attachments")

TEST FOR MTT INTERFERENCE
- 1 ml of a MTT solution (1 mg/mL) including 25±2 mg of the test item was incubated for 60min (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
- 25 ± 2 mg of the test item was added to 300 µl of deionised water and mixed. 330 µl of deionised water was used as control (blank). Both were incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2).
- 25 ± 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 38 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 37 min. After this incubation period the medium was changed, and the tissues were incubated for another 18 h. The complete post-incubation time was 41 h and 37 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 minutes 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 3 h 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):

41 h and 37 min

Number of replicates:

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

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

93.1

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 a colour change. Therefore, an additional test with freeze-killed tissues was necessary.

TEST FOR COLOUR INTERFERENCE
The test item did not change colour when mixed with deionised water or isopropanol. No colouring was detectable by unaided eye-assessment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of EpiDermTM for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the ten proficiency substances 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 was ≥ 0.8 and ≤ 2.8 (1.584) in accordance with OECD TG 439.
- the viability of the tissue replicates treated with the positive control was ≤ 20% (4.87).
- the standard deviation calculated from 3 identically treated replicates was ≤ 18 (0.0 - 4.5).
- 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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in the described EpiDerm study and under the experimental conditions reported, the test item sodium 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.