m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The study was performed for the assessment of the skin irritancy of the test item Bayscript Yell ow GGN with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS®.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

EC Number:

275-602-1

EC Name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Cas Number:

71550-21-5

Molecular formula:

C27H24N6O9S2.2C4H11NO2

IUPAC Name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Details on test material:

Test item: Bayscript Gelb GGN
Test item identity (including alternative names): Bayscript Gelb GGN Benzenesulfonic acid, 3,3’-(carbonylbis(imino(3-methoxy- 4,1-phenylene)-2,1-diazenediyl))bis-, compd. with 2,2’-iminobis(ethanol) (1:2)
CAS Number: 71550-21-5
Appearance: Russet colored crystaline solid

Specific details on test material used for the study:

Test item: Bayscript Yellow GGN
Chemical name: Benzenesulfonic acid, 3,3' -( carbonylbis(imino(3-methoxy-4, 1-
phenylene)-2, 1-diazenediyl)bis-, compd. with 2,2'iminobis(ethanol) (1 :2)
CAS number: 71550-21-5
Molecular mass: 850,9 g/mol
pH-value, diluted 1:9 in water: 4.6
Content: 89.6%
Appearance: solid, crystalline, russet

In vitro test system

Test system:

human skin model

Source species:

other: artificial 3D-Skin model

Cell type:

other: artificial 3D-Skin model (reconstructed human epidermis)

Cell source:

other: reconstructed human epidermis model epiCS® (CellSystems, Troisdorf, Germany)

Source strain:

other: artificial 3D-Skin model

Details on animal used as source of test system:

not applicable: artificial 3D-Skin model

Justification for test system used:

The model used for this study has a functional stratum corneum with an underlying layer of living cells as recommended by the test guidelines (e.g. OECD Test guideline 439).

Vehicle:

other: Test item was used undiluted.

Details on test system:

Test System/ Study Design
The model used for this study has a functional stratum corneum with an underlying layer of living cells as recommended by the test guidelines. The barrier function of the stratum corneum is adequate, as has been shown by the supplier.
The viability of the living cells in the model must be sufficiently high to discriminate well between the positive and negative control substances. Cell viability is measured by the amount of MTT reduction, i.e. an OD value, following exposure to the negative control substance or the test item.

Methods and Parameters
Reconstructed tissues
The experiment was carried out on the reconstructed human epidermis model epiCS® (CellSystems, Troisdorf, Germany). The tissue equivalents were shipped in 24 well cell culture plates on agarose supplemented with maintenance medium (Kit contents epiCS® CellSystems, Cat.-No.CS-1001). Inserts were of 0.6 cm² size.

Adaptation to cell culture conditions
Inserts with epiCS® reconstructed human epidermis (0.6 cm2) were packed under sterile conditions and were shipped refrigerated on supplemented agarose. Upon arrival, 6 well culture plates were pre-filled with 1 ml of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5% C02, 37°C, max humidity) afterwards for at least 6 hours before use.

4.4.3 Environmental conditions
The environmental conditions in the incubator were standardized as follows:
Incubator temperature 37 +/- 2°C
CO2 gas concentration 5%
Humidity maximum
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door. However, these deviations had no effect on the course or outcome of the study. All Incubation steps were performed in a CO2 atmosphere incubator.

Test item formulation
Test item was used undiluted.

Application of the test item and incubation
The epiCS® inserts were exposed to 30 mg of the test item (plus 30 J.tl 0.9% NaCl to moisten and ensure good contact with the epidermis surface) for 20 min. (RT, three inserts). 0.9% NaCl and 5% SDS (each 30 J.tl) treated epidermal models were used as negative and positive controls, respectively (determination in triplicates). A piece of mesh was used as a spreading aid for the test item.

Determination of cell viability (MTT assay)
After the exposure to the test item the inserts were washed carefully in PBS. After a post - treatment incubation period of 42 h of the rinsed tissue in the incubator a MTT assay was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µl ofMTT solution (37°C, 1 mg/ml in MTT-assay medium, delivered by Cell Systems®). The tissues were incubated for about 3 hours under cell culture conditions (5% CO2, 37°C, max humidity). The extraction of blue formazan was performed in isopropanol (24 well plates, 2 ml per insert) on a vertical shaker (2 hours). The concentration of formazan was measured by determining the OD of the isopropanol-extracts in duplicates at 570 nm in an automatic reader.
The MTT reduction assay is the most frequently used assay for the determination of cell viability. The assay depends on the intracellular capacity ofliving cells to chemically reduce the yellow 3- [4,5-Dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) to blue formazan crystals. The test has shown to give accurate and reproducible results in various laboratories and has practically been modified for accurate analysis of cell viability in three dimensional skin models.

Control samples:

other: Negative control: NaCl 0.9%, Positive control: 5% SDS solution

Amount/concentration applied:

The epiCS® inserts were exposed to 30 mg of the test item (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface) for 20 min. (RT, three inserts).

Duration of treatment / exposure:

The epiCS® inserts were exposed to 30 mg of the test item (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface) for 20 min. (RT, three inserts).

Duration of post-treatment incubation (if applicable):

After the exposure to the test item the inserts were washed carefully in PBS. After a post - treatment incubation period of 42 h of the rinsed tissue in the incubator a MTT assay was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µl ofMTT solution (37°C, 1 mg/ml in MTT-assay medium, delivered by Cell Systems®). The tissues were incubated for about 3 hours under cell culture conditions (5% CO2, 37°C, max humidity).

Number of replicates:

3 replicates.

Test animals

Species:

other: not applicable: artificial 3D-Skin model

Strain:

other: not applicable: artificial 3D-Skin model

Test system

Type of coverage:

other: not applicable: artificial 3D-Skin model

Preparation of test site:

other: not applicable: artificial 3D-Skin model

Vehicle:

other: not applicable: artificial 3D-Skin model

Amount / concentration applied:

not applicable: artificial 3D-Skin model

Duration of treatment / exposure:

not applicable: artificial 3D-Skin model

Observation period:

not applicable: artificial 3D-Skin model

Number of animals:

not applicable: artificial 3D-Skin model

Details on study design:

not applicable: artificial 3D-Skin model

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Bayscript Yellow GGN

Value:

97.3

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:

All acceptance criteria were met.
The test item Bayscript Yellow GGN did not show a significant impact on cell viability and is thus identified as non-irritant substance in this test model.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Bayscript Yellow GGN did not show a significant impact on cell viability and is thus identified as non-irritant substance in this test model.

Executive summary:

The skin irritancy of the test item Bayscript Yellow GGN was assessed with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS®.

The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.

Cell viability was measured in a photometer by the amount ofMTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100%

The results of the concurrent negative control (NC, 0.9% NaCl) and positive control (PC, 5% SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.

The following value of cell viability was recorded for the test item: 97 % (rounded).

In conclusion the results of the assay used show no skin irritant properties of the test item Bayscript Yell ow GGN and thus, the test item requires no classification according to UN GHS (Category 2 or Category 1).