Disodium 4,5-dihydroxybenzene-1,3-disulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 - 23 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovenska Narodna Akreditacna Sluzba (SNAS), Bratislava, Slovenska republika

Test material

Test material information

1

Reference substance name:

Disodium 4,5-dihydroxybenzene-1,3-disulphonate

EC Number:

205-741-5

EC Name:

Disodium 4,5-dihydroxybenzene-1,3-disulphonate

Cas Number:

149-45-1

Molecular formula:

C6H6O8S2.2Na

IUPAC Name:

disodium 4,5-dihydroxybenzene-1,3-disulphonate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200, MatTek)
- Tissue batch number: 28614

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with Dulbecco’s phosphate buffered saline (DPBS) to remove any residual test material (20 times).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: plate spectrophotometer
- Wavelength: 540 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 2.045 ± 0.114 (acceptance criteria: 1.0 - 3.0).
- Barrier function: According to the Supplier`s Data Sheet, the ET50 value was determined to be 5.06 h (Lower acceptance limit ET50 = 4.77 h, Upper acceptance limit ET50 = 8.72 h).
- Morphology: Tissue viability and barrier function test were in the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of afunctional stratum corneum, a viable basal layer and intermediate spinous and granular layers
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.

NUMBER OF REPLICATE TISSUES: 2 replicates for each treatment condition (3 min and 1 h experiment)

TEST FOR DIRECT MTT REDUCTION
To identify if the test item directly reduces MTT, this test was performed:
The test item in the amount of 25 mg was added to 1 mL of the MTT medium and was incubated in the dark at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 for 60 min. Untreated MTT medium was used as control. The color of treated MTT remained unchanged, so that the test items didn’t reduce of MTT directly.

ASSESSMENT OF COLORED OR STAINING MATERIALS
Some non-colored test materials may change into colored materials in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility was performed.
For this purpose, the test item in the amount of 25 mg was added into 0.3 mL of purified water. The mixture was incubated in the incubator at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 60 min. At the end of the exposure time the presence of the staining was evaluated. The colour of mixture was unchanged and the test item had not the potential to stain tissue.

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

ACCETANCE CRITERIA
The assay was considered valid if the following criteria were met:
Negative control (NC): The absolute optical density (OD) of the water treated negative control tissues in the MTT-test is an indicator of tissue viability obtained under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the mean of NC is OD ≥ 0.8.
Positive control (PC): One hour exposure of the positive control must reveal a mean tissue viability less than 15%.
Coefficient of variation (CV): In the range between 20% and 100% viability the CV is an additional acceptance criterion. It should not exceed 0.3.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean tissue viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg test item + 25 µL water

NEGATIVE CONTROL
- Amount applied: 50 µL aqua pro injectione

POSITIVE CONTROL
- Amount applied: 50 µL potassium hydroxide (KOH)
- Concentration: 8 N

Duration of treatment / exposure:

3 min and 1 h

Number of replicates:

2 tissue replicates per treatment

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean relative tissue viability from 2 tissues

Run / experiment:

3 min experiment

Value:

74.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean relative tissue viability from 2 tissues

Run / experiment:

1 h experiment

Value:

99.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Direct-MTT reduction:
The test substance showed non specific MTT-reducing potential.

- Colour interference with MTT:
The test substance showed no colour interference potential.

ACCEPTANCE OF RESULTS:
The tissue viability met the acceptance criterion (mean OD of negative control was 1.796 and 1.429, respectively).
The viability of culture treated by positive control 8N KOH was 14.2% and 6.1%, respectively. The positive control met the acceptance criterion: mean tissue viability less than 15% after one hour exposure.

Any other information on results incl. tables

Table 2: Skin corrosivity potential of the test item after 3 min and 1 h exposure in human skin model test EpiDermTM

	3 min		1 hour
Test Item	OD mean	viability mean (%)	OD mean	viability mean (%)	in vitro prediction
Negative Control (H2O)	1.796	100.0	1.429	100.0	non-corrosive
Positive Control (8 N KOH)	0.254	14.2	0.087	6.1	corrosive
Test item	1.335	74.3	1.420	99.3	non corrosive

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive according to OECD 431

Conclusions:

Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiDerm™ model, but no prediction on the skin irritation potential can be made and additional testing should be conducted for classification and labelling purposes.