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Toxicological information

Skin sensitisation

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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
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
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- 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: Crl: NMRI BR (SPF-bred)
- Source: Charles River Germany Sulzfeld, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: 28-32 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:
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 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
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 count indices, has just been exceeded in the high dose group. This increase is of statistical significance. Cell count index (test item concentration): 1.00 (0 %) / 0.88 (2 %) / 1.11 (10 %) / 1.42 (50 %). The EC 1.4 value calculated is 47.42 % for this test item.
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

It has to be clarified that the “positive level” mentioned above is exclusively defined for the NMRI outbreed mice used for this study. Such positive limits have to be calculated for each strain of mice individually.

The mice showed a clear increase in the weights of the draining lymph nodes in the mid and the high dose group, which are of statistical significance, compared to control animals (weight index: 1.0 (0 %), 0.95 (2 %), 1.36 (10 %), 1.46 (50 %)).

Statistically significant increases of the ear swelling and ear weights were determined for the high dose group animals compared to vehicle treated animals (ear swelling: day 1 = 17.42 (0 %) / 16.92 (2 %) / 17.42 (10 %) / 17.75 (50 %); day 4 = 17.75 (0 %) / 17.42 (2 %) / 18.58 (10 %) / 42.00 (50 %); Index day 4 = 1.00 (0 %) / 0.98 (2 %) / 1.05 (10 %) / 2.37 (50 %) ear weight: day 4 = 11.99 (0 %) / 12.21 (2 %) / 12.94 (10 %) / 27.61 (50 %); Index day 4 = 1.00 (0 %) / 1.02 (2 %) / 1.08 (10 %) / 2.30 (50 %)).

The increases in the high dose group may partly be due to left-overs of the test item, which became a very viscous formulation at this concentration (50 %). A thin layer, unremovable from animal hair and wood shavings had been recognized on the ears before sacrifizing the animals. In addition a hairless area around the base of the ears was observed.

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 the substance formulated in acetone/olive oil at 0 % (vehicle control), 2 %, 10 % and 50 %. Compared to controls the mice showed a significant increase in the weights and in the cell counts of the draining lymph nodes. For the cell counts the “positive level” of 1.4 has just been exceeded in the high (50 %) dose group.

Statistically significant increases of ear swelling and ear weights were also determined for high dose animals. An increase in these parameters does point to an acute irritant (inflammatory) response, which may partly be due to left-overs of the test item, which became a very viscous formulation at the high dose concentration (50 %). A thin layer, unremovable from animal hair and wood shavings had been recognized on the ears before sacrificing the animals. In addition a hairless area around the base of the ears was observed.

Due to the irritant property of the test item at the highest concentration it is not quite clear whether the cell proliferation is induced by a non-specific (inflammatory) reaction or a specific (sensitizing) reaction.

Summarizing, a sensitizing potential cannot be excluded by the data presented here. The concentration of 10 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization. The EC 1.4 value calculated was 47.42 %.