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

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011
Reference Type:
other:
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2010) - modified LLNA (IMDS): Measurement of cell counts instead of radioactive labelling. 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 lymph node 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: 7 weeks
- Weight at study initiation: 27-33 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:
acetone/olive oil (4:1 v/v)
Concentration:
0 (vehicle control), 2, 10, 50 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated once at day 1 of the study in acetone/olive oil. The formulation was 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 the experiences with the test system and the toxic properties of the test substance. For negative control a dose group treated only with the vehicle acetone /olive oil in the above described manner was used.
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 shows a clear increase in the stimulation indices for cell counts compared to control animals, which is of statistical significance. The “positive level”, which is 1.4 for cell count indices has clearly been exceeded.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Cell counts were increased in all dose groups, with statistical significance in the low and the mid dose group. The "positive level", which is 1.4 for the cell count index, has been exceeded in all dose groups. Cell count index (test item concentration): 1.00 (0 %) / 1.47 (2 %) / 2.19 (10 %) / 1.66 (50 %).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labeling

Any other information on results incl. tables

Compared to vehicle treated animals there were clear increases regarding the weights of the draining lymph nodes in the mid and the high dose group: weight index 1.00 (0 %) / 1.22 (2 %) / 1.79 (10 %) / 1.60 (50 %).
The “positive level” of ear swelling has been exceeded in the high dose group:
ear swelling day 1 = 17.58 (0 %) / 17.50 (2 %) / 17.42 (10 %) / 17.50 (50 %); day 4 = 18.00 (0 %) / 18.58 (2 %) / 18.50 (10 %) / 23.50 (50 %); Index day 4 = 1.00 (0 %) / 1.03 (2 %) / 1.03 (10 %) / 1.31 (50 %)
A significant increase compared to vehicle treated animals regarding ear weights was detected in the low and the high dose group: ear weight day 4 = 12.44 (0 %) / 13.79 (2 %) / 13.35 (10 %) / 15.74 (50 %); Index day 4 = 1.00 (0 %) / 1.11 (2 %) / 1.07 (10 %) / 1.27 (50 %).
The body weights of the animals were not affected by any treatment. This study does point to a non-specific (irritant) and 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 concentrations of 0 % (vehicle control), 2 %, 10 % and 50 %. The study does point to a non-specific (irritant) and to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell counts of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the formulated test item. Although it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 2 %.