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:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- 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 lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Stability under test conditions: The stability of the test item in the vehicle was analytically verified for up to 4 days.
The test item formulations in the vehicle were visually described as solutions.

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdWin:NMRI (SPF)
- Source: Harlan Nederland, 5960 AD Horst, The Netherlands
- Age at study initiation: 7 weeks
- Weight at study initiation: 26 - 32 g
- Housing: single housing in conventional Makrolon Type III cages
- Diet: Provimi Kliba SA 3883 maintenance diet for rats and mice, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 70
- Air changes (per hr): 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), 10, 30, 100 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated once before application in A/OO. 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 and positive control a dose group treated only with the vehicle A/OO and one treated with hexyl cinnamic aldehyde in A/OO, respectively, 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 is 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
The test is positive if the stimulation index exceeds 1.4 for cell count indices; for ear swelling if the "positive level" of 0.02 mm increase is reached. These levels are exclusively defined for the NMRI outbreed mice used in this study.
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 heterogenous (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 differentes 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:
After treatment with Alpha Hexyl Cinnamic Aldehyde mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to control animals, which are of statistical significance.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The “positive level”, which is 1.4 for cell counts has been exceeded in all dose groups. This increase is of statistical significance. Cell count index (test item concentration): 1.00 (0 %) / 2.31 (10 %) / 2.45 (30 %) / 2.90 (100 %). 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 10 %.
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

This 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 test substance.

Compared to vehicle treated animals there were clear increases regarding the weights of the draining lymph nodes and the cell counts, which are of statistical significance in all dose groups. The "positive level" of index 1.4 for the cell counts has been exceeded in all dose groups.
(Weight index: 1.0 (0%), 2.07 (10%), 2.41 (30%), 2.18 (100%); Cell count index: 1.00 (0 %) / 2.31 (10 %) / 2.45 (30 %) / 2.90 (100 %).)

The "positive level" of ear swelling which is 2x 10exp-2 mm increase, i.e. about 10 % of the control values, has been exceeded in all dose groups. These changes are of statistical significance.
(Ear swelling: day 1 = 17.17 (0 %) / 17.25 (10 %) / 17.25 (30 %) / 17.75 (100 %); day 4 = 17.50 (0 %) / 24.50 (10 %) / 26.42 (30 %) / 27.17 (100 %); Index day 4 = 1.00 (0 %) / 1.4 (10 %) / 1.51 (30 %) / 1.55 (100 %).)

A significant increase compared to vehicle treated animals regarding ear weights was detected in all dose groups.
(Ear weight = 11.78 (0 %) / 16.92 (10 %) / 18.28 (30 %) / 18.18 (100 %); Index day 4 = 1.00 (0 %), 1.44 (10 %), 1.55 (30 %), 1.54 (100 %)).

Differentiation indices (DI), which is the quotient of the relative lymph node reaction divided by the relative acute skin reaction was < 1 for all concentrations tested, i.e. 0.82, 0.71 and 0.86 (for calculation of DI see: Homey et.al., Toxicol. and Appl. Pharmacol. 153, 1998, 83 -94; Vohr et.al. Arch. Toxicol. 73, 2000, 501 -509). These DI values point to an irritating potential of the test item. However, such an irritant property could also be combined with a skin sensitizing potential of a test compound. Thus, a skin sensitizing property cannot be excluded.

The body weights of the animals were not affected by any treatment.

 

Applicant's summary and conclusion

Executive summary:

A modified LLNA (IMDS; OECD TG 429) was performed on 6 female NMRI mice per dose group using test substance of 0 % (vehicle control), 10 %, 30 % and 100 %.

Compared to vehicle treated animals clear increases regarding the weights of the draining lymph nodes and the cell counts were seen. The "positive level" of index 1.4 for the cell counts has been exceeded in all dose groups.

The "positive level" of ear swelling has also been significantly exceeded and a significant increase regarding ear weights was detected. These changes were of statistical significance in all dose groups. An increase in these parameters would point to an acute irritating (inflammatory) response. However, such an irritant property could also be combined with a skin sensitizing potential of a test compound. Thus, a skin sensitizing property cannot be excluded.

Also 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 10%.

Summarizing, the study does point to a non-specific (irritant) and to a specific immunostimulating (sensitizing) potential of the test item.