disodium 4,4'-bis[(prop-2-enoyl)amino][1,1'-biphenyl]-2,2'-disulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 - 22 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

Adopted in 2019

Deviations:

yes

Remarks:

The demonstration of appropriate morphology by vendor is missing.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accreditation Service, Bratislava, Slovak Republic

Test material

Test material information

Constituent 1

Reference substance name:

disodium 4-[(1-hydroxyprop-2-en-1-yl)amino]-4'-(prop-2-enamido)-[1,1'-biphenyl]-2,2'-disulfonate

EC Number:

812-549-0

Cas Number:

881539-89-5

Molecular formula:

C18H14N2Na2O8S2

IUPAC Name:

disodium 4-[(1-hydroxyprop-2-en-1-yl)amino]-4'-(prop-2-enamido)-[1,1'-biphenyl]-2,2'-disulfonate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed human epidermal (RhE) model EpiDerm™ SIT (EPI-200), MatTek In Vitro Life Science Laboratories, Slovak Republic

Justification for test system used:

The test item is applied topically to a three-dimensional human reconstructed epidermis (RhE) model, comprised of non-transformed human-derived epidermal keratinocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers and a multilayered stratum corneum containing intracellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. Irritant chemicals are identified by their ability to decrease cell viability (as determined by using the MTT reduction assay) below defined threshold levels. The EpiDerm™ SIT allows discrimination between substances requiring classification and labeling (EU GHS Cat. 1 or 2) and non-irritants according to the EU and GHS classification system.

Vehicle:

unchanged (no vehicle)

Remarks:

Shortly before application of the test item, the tissue surface was moistened with 25 µL sterile DPBS to improve contact of the tissue surface with the test item.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ SIT (EPI-200), MatTek In Vitro Life Science Laboratories, Slovak Republic
- Tissue batch number(s): Lot no. 30836, Keratinocyte strain: 00267
- Production date: 20 Nov 2019 (Certificate of Analysis from MatTek)
- Delivery date: 18 Nov 2019
- Date of initiation of testing: 19 Nov 2019

Comment on the production date and experimental starting date: The tissue supplier states that the production date for the tissues produced by MatTek IVLSL is Monday. The lab performs preQC and if the tissues do not fulfil the quality control criteria the tissues are not shipped to the customers. To obtain the real life data for the tissues shipped to the customers, MatTek mimics the shipment conditions and therefore performs the quality controls on Tuesdays with quality control measurements on Wednesdays. Therefore the certificates of analysis for the tissues are issued on Wednesdays. This is the standard procedure for RhE tissues currently produced at MatTek IVLSL.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment/ exposure: 35 min at 37 °C + 25 min at room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed 15 times, then submerged 3 times in 150 mL Dulbecco's Phosphate-Buffered Saline (DPBS). Finally, the tissues were rinsed once from inside and once from outside with sterile DPBS and each insert was blotted on sterile blotting paper and transferred to new 6-well plates prefilled with 0.9 mL of fresh assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT/ EXPOSURE
- MTT concentration: 1 mg/mL; 0.3 mL/well of MTT solution
- Incubation time: 3 h
- Spectrophotometer: Dynex MRX II
- Wavelength: 540 nm
- Filter: without a reference filter

FUNCTIONAL MODEL CONDITIONS
- Viability: The quality of the RhE tissue was assessed by an MTT cell viability test (4 h, n=3). The determined OD (540 - 570 nm) was 2.002 ± 0.169 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) following application of 100 µL 1% Triton X-100. Four timepoints (4, 6, 8 10 h exposure) were evaluated. The ET-50 value was determined to be 4.85 h (acceptance criteria: 4.77 - 8.72 h).
- Contamination: The cells used to produce the RhE tissue were screened for the presence of HIV-1 virus, Hepatitis B virus, Hepatitis C virus, bacteria, yeast, and other fungi. No contamination was detected.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Test for interference of test substance with MTT endpoint:
Test substances may interfere with the MTT endpoint, if they are coloured and/or able to directly reduce MTT and at the same time are present in the tissues when the MTT viability test is performed. Some non-coloured test substances may change into coloured material under wet or aqueous conditions and thus stain tissues during the 60 min exposure period. To identify these possible interferences, a functional check was performed.
The test substance in amount of 25 mg was added into 0.3 mL of purified water. The mixture was incubated in glass test tube 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.
Then, 25 mg of the test substance was added to 1 mL of the MTT medium and incubated in the incubator at 37 ± 1 °C in a humidified atmosphere of 5 ± 1% CO2 in air for 60 min. Untreated MTT medium was used as control.
The colour of MTT solution remained unchanged and it was concluded that the test substance did not reduce MTT directly.
An additional functional check was not performed.

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

PREDICTION MODEL/ DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 60 min exposure is ≤ 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 min exposure is > 50%.

DEMONSTRATION OF TECHNICAL PROFICIENCY TESTING
Demonstration of proficiency is stated in amendment No. 1 to the final report. The laboratory demonstrates technical proficiency by the non-classified substances (UN GHS no Category) and classified substances (UN GHS Category 2) indicated by the OECD 439 (version 25 Jul 2015) in vitro skin irritation test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- The white powder was applied unchanged using a spoon. However, in accordance with OECD TG 439, shortly before application of the test item, the tissue surface was moistened with 25 µL sterile DPBS to improve contact of the tissue surface with the test item.

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

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

Duration of treatment / exposure:

60 min (35 min at 37 °C and 25 min at RT)

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

Triplicates for each treatment and control group.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min exposure

Value:

87.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT: The test substance did not change the colour, when mixed with deionised water, and passed the colour interference pre-test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the tissue viability met the acceptance criterion: mean OD of negative control was 1.334.
- Acceptance criteria met for positive control: Yes, the positive control met the acceptance criterion: mean tissue viability was 4.2% (i.e. < 20%).
- Acceptance criteria met for variability between replicate measurements: Yes, SD calculated from individual % tissue viabilities of three identically treated replicates was < 18%.
See Table 1 under "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1. Skin irritation potential of AF-959 after 60-minute exposure in reconstructed human epidermal model EpiDerm™ SIT

Test item	Mean of OD	SD of OD	Mean of cell viabilities (%)	SD of cell viabilities	CV (%)	Classification
Negative control (DPBS)	1.334	0.025	100	1.91	1.91	non-irritant
Positive control (5% SDS)	0.056	0.002	4.2	0.15	3.57	irritant
Test substance (AF-959)	1.162	0.054	87.1	4.07	4.68	non-irritant

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

Under the conditions of the present study, AF-959 is considered to be non-irritant to skin (GHS No Category).