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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 days
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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 %
Specific details on test material used for the study:
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.)
Source strain:
other: SkinEthic Laboratories, Lyon, France.
Details on animal used as source of test system:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-040).
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).
Vehicle:
other: Milli-Q water
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:
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.