N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

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:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

EC Number:

264-439-1

EC Name:

N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

Cas Number:

63741-10-6

Molecular formula:

C17H17N4O2Cl3

IUPAC Name:

N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

Test material form:

solid: particulate/powder

Details on test material:

Anthraquinone dye, blue powder

Specific details on test material used for the study:

The batch of Disperse Brown 27 (4852-1501) tested was a brown powder with a purity of 99.7 %

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-036

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Details on test system:

This model is a three-dimensional human epidermis model, which consists of adult humanderived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (10.3 to 12.7 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

Duration of treatment / exposure:

The test consists of topical application of Disperse Brown 27 on the skin tissue for 15 minutes. The tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Duration of post-treatment incubation (if applicable):

Post-treatment incubation peiod was 42 hours.

Number of replicates:

Three sets of replicates, one each for Disperse Brown 27, positive and negative control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 113

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

it is concluded that this test is valid and that Disperse Brown 27 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

Disperse Brown 27 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Disperse Brown 27 did not interact with the MTT endpoint.

The mean tissue viability obtained after 15 ± 0.5 minutes treatment with Disperse Brown 27 compared to the negative control tissues is as follows. Skin irritation is expressed asvthe remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Disperse Brown 27 compared to the negative control tissues was 113%. Since the mean relative tissue viability for Disperse Brown 27 was above 50% Disperse Brown 27 is considered to be non-irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 5.3%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 11% or less, indicating that the test system functioned properly.