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Description of key information

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:
other: acccording to guideline and GLP and additional discriminating measurements sensitizing and irritationg properties
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The modification refer to the measurement of cell proliferation by cell counting instead of radioactive labelling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immun system
Principles of method if other than guideline:
Modified LLNA (IMDS = Integrated Model for the Differentiation of Skin Reactions): The modification refer to the measurement of cell proliferation by cell counting instead of radioactive labelling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immun system, as also recommended in the update of OECD TG 429.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5 weeks
- Weight at study initiation: 26-33 g
- Housing: during the study individually
- Diet ad libitum
- Water. ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:
methyl ethyl ketone
Concentration:
0% (vehicle control), 2 %, 10 %, 50 %, and positve control 30 % alpha hexyl cinnamic aldehyde in DMF
No. of animals per dose:
6
Details on study design:
The test item in the formulation, the positive control in the formulation or the vehicle were applied epicutaneousely onto the dorsal part of both ears.of the animals. This treatment was repeated on three consecutive days. The volume administered was 25 µl/ear. The used concentrations were based on the experience with this test system and the properties of the test item.
The animals were anaestetized by inhalation of carbon dioxid and sacrificed one day after the last application. The appropriate organs were then removed .Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline
Investigations:
-weight of lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell count of the subtancetreated lymp nodes by the vehicle treated ones
- ear swelling
- ear weight
-body weight
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group by one-way Analysis of Vartiance (ANOVA) when the variances are considered homogenous according to a homogenicity testig like Cochran's test. Alternatively, if the variances are considered to be heterogenous a non-parametric Kruskal-Wallis test has been used at significance levels of 5 %.. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99 % by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Sheffe's method, wich can be used for both equal and unequal sample sizes.
Parameter:
SI
Remarks:
Cell count index
Value:
1
Test group / Remarks:
Gr. 1 (Vehicle (MEK))
Remarks on result:
other: modified LLNA; mearurement of cell counts instead of radioactive labelling.
Parameter:
SI
Remarks:
Cell count index
Value:
0.77
Test group / Remarks:
Gr. 2 (2 %Nigrosin WLF (in MEK))
Remarks on result:
other: modified LLNA; mearurement of cell counts instead of radioactive labelling.
Parameter:
SI
Remarks:
Cell count index
Value:
0.78
Test group / Remarks:
Gr. 3 (10% Nigrosin WLF (in MEK))
Remarks on result:
other: modified LLNA; mearurement of cell counts instead of radioactive labelling.
Parameter:
SI
Remarks:
Cell count index
Value:
0.74
Test group / Remarks:
Gr. 4 (50 %Nigrosin WLF (in MEK))
Remarks on result:
other: modified LLNA; mearurement of cell counts instead of radioactive labelling.
Parameter:
SI
Remarks:
Cell count index
Value:
2.19
Test group / Remarks:
Gr. 5 (30 % Alpha Hexyl Cinnamic Aldehyde (in MEK))
Remarks on result:
other: modified LLNA; mearurement of cell counts instead of radioactive labelling.

It has to be clarified that the positive levels are exclusively defined for the NMRI mice used for this study:

The positive level of ear swelllling which is 2x10 [exp -2] mm increase , i.e. about 10 % of the control values, has not been reached or exceeded in any dose group.of the test item whereas the respective value of the positive control has shown a statistical significant increase.

Body weights of the animals was not affected by any treatment.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Executive summary:

A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of formulation

containing 0%, 2 %, 10 %, or 50 % of the test item Nigrosin WLF dissolved in Methylethylketon (MEK) for 3 consecutive days

onto both ears of the animals. The study was conducted according to OECD TG No. 429 and No. 406.

Nigrosin WLF showed no sensitizing potential in the modified Local Lymph Node Assay (IMDS) in female NMRI mice after dermal application of up to and including a 50 % concentration. Additionally, no indication for a non-specific(irritant) activation by the test item was detected. The positive control hexyl cinnamic aldehyde (CAS-No. 101 -86 -0) was functional for each parameter (Vohr 2012)..

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

The modified Local Lymph Node Assay (IMDS) was performed in 2012 on 24 female NMRI mice of the strain HsdWin:NMRI (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item Nigrosin WLF in methylethylketon (MEK): 0% (vehicle control),.2 %, 20 %, 50 %. A concurrent control of 6 animals treated with 30% Alpha Hexyl Cinnamic Aldehyde in MEK was included. The study was conducted according to OECD Guidelines No. 429 and No. 406, EC Guideline 2004/73/EC (29th Adaptation of Guideline 67/548/EEC, B.42)/Health Effects Test Guideline and OPPTS 870.2600 (EPA)

The results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 50 % Nigrosin WLF in this test system. In conclusion, these results show that the test item Nigrosin WLF has no sensitizing potential in mice after dermal application of up to and including a 50 % concentration. No indication for a non-specific (irritant) activation was detected, too. Therefore, the concentration of 50 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization. These results are verified by the comparison with the results of the positive control group (Alpha Hexyl Cinnamic Aldehyde).


Migrated from Short description of key information:
The modified Local Lymph Node Assay (IMDS) was performed according to OECD Guidelines No. 429 and No. 406. The results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 50 % Nigrosin WLF in this test system.

Justification for selection of skin sensitisation endpoint:
There is only one study available .

Justification for classification or non-classification

Based on the available data no classification or labelling is required