Disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-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:

March 2016 - May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 03 N0vember 2017

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: yellow/brown powder
- Storage conditions: in refrigerator (2-8°C)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: All cells are derived from tissue obtained by MatTek Corporation from accredited institutions.

Source strain:

other: 00267

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm Skin Model consists of normal, 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 multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

MEDIA
- DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM medium, serum-free supplied by MatTek Corporation.
- MTT medium. MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 - 86 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.3°C). Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

ACCEPTABILITY OF THE ASSAY
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DATA EVALUATION AND STATISTICAL PROCEDURES
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: concurrent MTT reduction control

Amount/concentration applied:

29.1 to 44.7 mg of the solid test item

Duration of treatment / exposure:

Tissues were exposed for 3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

MTT-medium and tissues were incubated for 3 hours post exposure

Number of replicates:

2 replicates for 3 minute exposure and 2 replicates for 1 hour exposure

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean relative tissue viability obtained after 3-minute treatment with the test item compared to the negative control tissues.

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Milli-Q water

Positive controls validity:

valid

Remarks:

8N KOH

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean relative tissue viability obtained after 1-hour treatments with the test item compared to the negative control tissues.

Value:

81

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Milli-Q water

Positive controls validity:

valid

Remarks:

8N KOH

Other effects / acceptance of results:

The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

Controls:
Positive control: mean relative tissue viability of 10% after 3 minutes exposure.
Negative control: The absolute mean OD570 (optical density at 570 nm) was within the laboratory historical control data range.
Coefficient of variation between tissues replicates (range 20-100% viability): ≤ 7%, indicating that the test system functioned properly.

Effects:
Mean relative tissue viability for the test item was not below 50% after 3 minute exposure
Mean relative tissue viability for the test item was not below 15% after 1 hour exposure

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin corrosion test with Disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate, performed according to OECD/EC test guidelines, no corrosion was observed.

Executive summary:

An in vitro skin corrosion test was performed with Disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate using a human skin model (EpiDerm (EPI-200)) according to OECD/EC guidelines and GLP principles. The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 96% and 81%, respectively.Negative and positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Based on these results, 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonic acid is not classified for corrosive effects on the skin.