Registration Dossier

Administrative data

Description of key information

Test material is not a sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
44 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
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:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Two test item concentrations were tested for the pre-screen test, 25 and 40% with two female mice used for each concentration.
For the main study 4 experimental groups each consisting of 5 female CBA/J mice, 1 group was used for the vehicle (propylene glycol), the other groups were used for seperate concentrations of test material (5, 10, 25% etc)
Vehicle:
propylene glycol
Concentration:
0, 5, 10 and 25% concentration of test item.
No. of animals per dose:
For the main study 4 experimental groups each consisting of 5 female CBA/J mice, 1 group was used for the vehicle (propylene glycol), the other groups were used for seperate concentrations of test material (5, 10, 25% etc)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The SI values calculated for the positive control item concentrations 5, 10 and 25% were 1.4, 2.4 and 4.3 respectively. An EC3 value of 14,7% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years away were 13.4, 14.1, 17.3, 9.8, 17.8 and 18.0%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
The raw data, study plan and report from this study are kept in the Charles River Den Bosch archives. The test described above was performed in accordance with Charles River Den Bosch Standard Operating Procedures and the report was audited by the QA-unit.
Key result
Parameter:
SI
Value:
>= 1.7 - <= 2.1
Variability:
range of 0.4
Cellular proliferation data / Observations:
No erythema was observed in any of the animals. Blue test item remnants were present on the dorsal surface of the ears of all test item treated animals, but did not hamper the scoring of the skin reactions. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 431, 438 and 544 DPM, respectively. The mean DPM/animal value for the vehicle control group was 254 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.7, 1.7 and 2.1, respectively.
Interpretation of results:
GHS criteria not met
Conclusions:
Mean DPM/animal values for the experimental groups treated with test item concentration 5, 10 and 25% were 431, 438 and 533 DPM, respectively. The mean DPM/animal value for the vehicle control group was 254 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.7, 1.7 and 2.1, respectively.
Since there was no indication that the test time elicits a SI ≥ 3when tested up to 25%, Disperse Blue 359 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration what will give SI=3) (if any) exceeds 25%.
Executive summary:

Test item concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test item concentration of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Propylene glycol).

Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentration 5, 10 and 25% were 431, 438 and 533 DPM, respectively. The mean DPM/animal value for the vehicle control group was 254 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.7, 1.7 and 2.1, respectively.

Since there was no indication that the test time elicits a SI ≥ 3when tested up to 25%, Disperse Blue 359 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration what will give SI=3) (if any) exceeds 25%.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, Disperse Blue 359 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

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

Justification for classification or non-classification