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Administrative data

Description of key information

Direct Blue 199 ammonium salt was tested in the Local Lymph Node Assay (OECD 429, GLP) in form of a commercial aquaous formulation containing 19% of the colorant and 8% urea (BASF 2003).

It was dosed in the pure form (undiluted) or at a diluation of 10% or 30% in propylene glycol. Most solids can only applied at pastes of ca 25% content. The high concentration of 19% in water can only be achieved by the addition of 8% urea. Therefore 19% as highest concentration is seen as the highest attainable and therefore acceptable concentration for testing in the LLNA.

There was no increase in lymph node weight nor lymph node cell count. As a result, the substance was found to be a non skin sensitizer in the LLNA.

Experimental data from a guinea pig maximization test (OECD 406, GLP) is available for Direct Blue 199 as a mixed ammonium/sodium salt (BASF 1989). The test material had a purity of 83% and was applied at 1% and 10% for intradermal and epicutaneous induction, respectively. The challange concentration was 5% which was the highest non-irritating concentration. As no local reactions towards challenge exposure were observed, the substance was found to be non-skin sensitizing in the GPMT.

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
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)
Version / remarks:
non-radioactive version
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Composition:
19% CAS 94543-99-3
72% Water
8% Urea
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Age at the beginning of the study: about 8 weeks
Body weight range at day 0: 18.5 g - 22.2 g
The single housed animais were identif led by cage cards.
The animals were housed in fully air-conditioned rooms in which a central air-conditioning system ensured a temperature in the range of 20 - 24°C and a relative humidity in the range of 30 - 70%. The day/night rhythm was 12 h light and 12 h darkness.
Single housing in Makrolon type I cages.
Bedding: Granulat Typ 3/4 (staubfrei); SSNIFF
Type of diet: Kliba Labordiaet (Ratte-Maus-Hamster Haltungsdiaet), Provimi Kliba SA, Kaiseraugst, Switzerland ad libitum
Watering: Tap water ad libitum
Acclimatization period: At least 8 days before the first test substance application
Vehicle:
propylene glycol
Remarks:
for the high dose, the test material was used in the pure form.
Concentration:
Undiluted or 10% or 30% in propylene glycol
Each test animal was applied with 25 uL per ear of the respective test substance / test substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 uL per ear of the vehiole alone.

19% is the solubility limit. Higher concentrations are not possible.
No. of animals per dose:
6
Details on study design:
The test substance preparations were produced on a weight by weight (wlw) basis shortly before the application by stirring with a magnetic stirrer.
The homogeneity of the test substance preparations during application was provided by stirring with a magnetic stirrer.
Form of test substance preparations: The test substance was applied undiluted resp. as an emulsion for all applications.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Remarks:
Cell count
Value:
1.11
Test group / Remarks:
10% in propylene glycol (1.9% of the substance)
Parameter:
SI
Remarks:
cell count
Value:
0.97
Test group / Remarks:
30% in propylene glycol (5.7% of the substance)
Parameter:
SI
Remarks:
cell count
Value:
1.07
Test group / Remarks:
undiluted (19% of the test substance)
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.08
Test group / Remarks:
10% in propylene glycol
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.17
Test group / Remarks:
30% in propylene glycol
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.15
Test group / Remarks:
Undiluted (19% of the test substance)

Table 1: Individual values for the lymph node weights

Test Group Animal No. Lymph Node Weight [mg]
Individual Mean S.D.
  1 4.0    
  2 4.2    
1
vehicle propylene glycol
3 5.3 4.6 0.6
  4 5    
  5 4.9    
  6 4.0    
  7 4.3    
  8 4.0    
2
10% in propylene glycol
9
5.8
5.0 0.7
  10 5    
  11 5.5    
  12 5.1    
  13 5.3    
  14 4.3    
3
30% in propylene glycol
15 6.0
5.3 0.6
  16 5.3    
  17 5.3    
  18 5.8    
  19 6.5    
  20 5.5    
4
undiluted
21
4.2
5.2 0.9
  22 4.7    
  23 5.8    
  24 4.7    

Table 2: Individual counts for the cell counts

Test Group Animal No. Cell Count [Count/Lymph Node Pair]
Individual Mean s.d.
  1 7'062'000    
  2 8'380'000    
1
vehicle propylene glycol
3 9,538,000
7'968'333 1'179'450
  4 8,524,000    
  5 8'118'000    
  6 6'188'000    
  7 6'646'000    
  8 6'160'000    
2
10% in propylene glycol
9 10,930,000
8'858'667 1'994'323
  10 9,316,000    
  11 10'480'000    
  12 9'620'000    
  13 9'442'000    
  14 4'699'000    
3
30% in propylene glycol
15
7,558,000
7'733'500 1'695'198
  16 7,760,000    
  17 7'722'000    
  18 9'220'000    
  19 12'858'000    
  20 7'420'000    
4
undiluted
21
6,826,000
8'506'000 2'390'135
  22 6,246,000    
  23 9'222'000    
  24 8'464'000    

s.d. = standard deviation

Table 3: Ear weights

Test Group Treatment Mean S.D. Index 
1 vehicle propylene glycol 27.4 0.9 1.00
2 10% in propylene glycol 27.6 0.5 1.01
3 30% in propylene glycol 28.4 1.7 1.04
4 undiluted 27.9 1.3 1.02
Interpretation of results:
GHS criteria not met
Conclusions:
No signs of systemic toxicity were noticed. The test material (19% aquaeous formulation) did not induce a statistically significant and biologicaily relevant response of the auricular Iymph nodes when applied as 10% preparation in propylene glycol or applied as the undiluted test substance. The statisticaliy significant increase in lymph node weights of mice treated with the 30% test material preparation was not in congruency with the ceIl counts, which were not statisticaily significant at all concentrations. The ear weights did not indicate a significant Irritation of the ear skin by the test substance.
From the results of the study it is concluded, that the 19% aquaeous formulation does not have a skin sensitizing effect in the Murine Local Lymph Node Assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The stimulation indices in the LLNA (OECD 429) did not show a dose dependent increase. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008,as amended for the seventh time in Regulation (EC) No 2015/1221.