1-amino-4-(ethylamino)-9,10-dihydro-9,10-dioxoanthracene-2-carbonitrile

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:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Additional procedure for test items that induce both colour interference and reduction of MTT was not performed. Procedure is designed to avoid possible double correction, without correction the test item is clearly negative. Does not affect study outcome

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-amino-4-(ethylamino)-9,10-dihydro-9,10-dioxoanthracene-2-carbonitrile

EC Number:

263-606-6

EC Name:

1-amino-4-(ethylamino)-9,10-dihydro-9,10-dioxoanthracene-2-carbonitrile

Cas Number:

62570-50-7

Molecular formula:

C16H14N2O2

IUPAC Name:

1-amino-4-(ethylamino)-9,10-dioxo-9,10-dihydroanthracene-2-carbonitrile

Test material form:

solid: particulate/powder

Details on test material:

Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99 %

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small Model (0.38 cm² reconstructed epidermis of normal human keratinocyes grown on a collegen matrix for 13 days.)

Vehicle:

other: Milli-Q water

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

11.0-13.8 mg of test item was added to the appropriate tissue samples

Duration of treatment / exposure:

Exposure period of 15 ± 0.5 minutes, following this the tissues were washed with phosphate buffered saline to remove residual test item

Duration of post-treatment incubation (if applicable):

Tissues were incubated for 48 hours at 37 °C

Number of replicates:

15 test tissued used, consiting of 5 replicatex of 3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 ± 0.5 minutes

Value:

ca. 98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative man tissue validity obtained after 15 ± 0.5 minutes treatment with Disperse Blue 359 compared to the negative control tissues was 98 %. Since the mean relative tissue viability for Disperse Blue 359 was above 50 % after 15 ± 0.5 minutes treatment Disperse Blue 359 is considered to be a non-irritant.
The positive control had a mean cell viability of 6.2 % after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissue was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 10 %, indicating that the test system function properly.
Finally, it is concluded that this test is valid and that Disperse Blue 359 is a non-irritant in the in vitro skin irritation test under the experimental conditions described.

Executive summary:

Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99%. Skin tissue was moistened with 5µL of Milli-Q water and at least 10 mg of Disperse Blue 359 was applied directly on top of the tissue for 15 ± 0.5 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Disperse Blue 359 did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Disperse Blue 359 was 4.4 % of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

Furthermore, Disperse Blue 359 showed colour interference in aqueous solutions. In addition to the normal procedure, three tissue were treated with test item. Instead of MTT solution this tissue was incubated with assay medium. The non-specific colour by Disperse Blue 359 was 4.9 % of the negative control tissues. The OD of the tissue incubated with assay medium was subtracted from the ODs of the test item treated tissues incubated with MTT medium.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue validity obtained after 15 ± 0.5 minutes treatment with Disperse Blue 359 compared to the negative control tissues was 98 %. Since the mean relative tissue viability for Disperse Blue 359 was above 50 % after 15 ± 0.5 minutes treatment Disperse Blue 359 is considered to be a non-irritant.

The positive control had a mean cell viability of 6.2 % after 15 ± 0.5 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissue was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 10 %, indicating that the test system function properly.

Finally, it is concluded that this test is valid and that Disperse Blue 359 is a non-irritant in the in vitro skin irritation test under the experimental conditions described.