Registration Dossier

Administrative data

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: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2002) - modified LLNA (IMDS): Measurement of cell counts instead of radioactive labeling. In addition, measurements of ear swelling and ear weights were done to discriminate the irritating potential from the sensitizing potential of the test substance.
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd Win: NMRI (SPF-bred)
- Source: Harlan Nederland, 5960 AD Horst, Netherland
- Age at study initiation: 8 weeks
- Weight at study initiation: 26-32 g
- Housing: During the study period the animals were single-housed in Makrolon type II cages.
- Diet and water: ad libitum
- Acclimation period: at least 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40-70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0 (vehicle control), 25, 50, 100 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the positive control in the formulation or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µL/ear. The used concentrations were based on experiences with the test system and the known properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of lymph nodes
- cell counts in lymph nodes
- stimulation index (calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones)
- ear swelling
- ear weight
- body weights
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 a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogeneous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) 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 Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

Positive control results:
Alpha hexyl cinnamic aldehyde, showed a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The "positive level", which is 1.4 for the cell count index, was never reached or exceeded in any dose group. Cell count index (test item concentration): 1.00 (0 %) / 1.16 (25 %) / 0.89 (50 %) / 0.92 (100 %).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labelling

Any other information on results incl. tables

Compared to vehicle-treated animals, also none of the other parameters measured in the substance-treated groups, i.e. weights of the draining lymph nodes, ear weights and ear swelling, reached or exceeded the "positive levels" defined for this assay. 
weight index: 1.00 (0 %) / 1.19 (25 %) / 1.00 (50 %) / 1.02 (10 %)
ear swelling: day 1 = 17.67 (0 %) / 17.75 (25 %) / 17.58 (50 %) / 17.33 (100 %); day 4 = 18.42 (0 %) / 18.92 (25 %) / 18.58 (50 %) / 18.92 (100 %); Index day 4 = 1.00 (0 %) / 1.03 (25 %) / 1.01 (50 %) / 1.03 (100 %)
ear weight: day 4 = 12.70 (0 %) / 12.38 (25 %) / 11.87 (50 %) / 12.29 (100 %); Index day 4 = 1.00 (0 %) / 0.98 (25 %) / 0.93 (50 %) / 0.97 (100 %).
The body weights of the animals were not affected by any treatment.
By comparing the specific immune reaction induced by the test item in the draining lymph nodes (cell counts / weights) with the immediate unspecific acute skin reaction (ear swelling / ear weight) it is possible to discriminate the irritant potential from the sensitizing potential of the compound tested.
This study does neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item.

Applicant's summary and conclusion

Executive summary:

A modified LLNA (IMDS) according to OECD TG 429 was conducted on 6 female NMRI mice per dose group with epicutaneously applied test substance of 0 % (vehicle control), 25 %, 50 % and 100 %. The study does neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. Therefore, the concentration of 100 % turned out to be the NOEL for the parameters investigated (weight and cell counts of the draining lymph nodes, ear swelling, ear weight) with respect to skin sensitization.