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Description of key information

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.

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

Disperse Blue 359 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.7 after 240 minutes of treatment. Since Disperse Blue 359 induced an IVIS ≤3, no classification is required for eye irritation or serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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
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%.
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
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
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes
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%.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -‘s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in a physiological saline in a suitable container under cooled conditions.The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from physiological saline and holding them in the light. Those exhibiting defects were discarded.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
318-391 mg of Disperse Blue 359
Duration of treatment / exposure:
Corneas were first incubated in cMEM for a minimum of 1 hour at 32 ± 1 °C, the corneas were then incubated with the tests material in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.
Number of animals or in vitro replicates:
9 samples in total, 3 replicates each for Disperse Blue 359, the positive control and the negative control.
Irritation parameter:
in vitro irritation score
Value:
ca. -0.7
Vehicle controls validity:
not specified
Negative controls validity:
valid
Remarks:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
Positive controls validity:
valid
Remarks:
The mean in vitro irritancy score of the positive control was 159 and within two standard deviations of the current historical positive control mean. It was therefore concluded that test conditions were adequate and that test system functioned properly.
Remarks on result:
no indication of irritation

The individual in vitro irritancy scores for the negative controls ranged from -0.8 to -0.9. The individual positive control in vitro irritancy scores ranged from 140-187. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with Disperse Blue 359 showed opacity values ranging from -1.2 to -0.4 and permeability values ranging from 0 to 0.0037. The corneas were clear were clear after the 240 minutes of treatment with Disperse Blue 359. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.2 to 0.2 after 240 minutes of treatment with Disperse Blue 359.

Interpretation of results:
GHS criteria not met
Conclusions:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 159 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Disperse Blue 359 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.7 after 240 minutes of treatment. Since Disperse Blue 359 induced an IVIS ≤3, no classification is required for eye irritation or serious eye damage.
Executive summary:

Study describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Disperse Blue 359 was tested through the topical application for approximately 240 minutes. Procedures were based on the most recent OECD guideline. Since no workable suspension of test material in physiological saline solution could be obtained, the test item was used as delivered and added pure on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 159 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Disperse Blue 359 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.7 after 240 minutes of treatment. Since Disperse Blue 359 induced an IVIS ≤3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification