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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:
weight of evidence
Study period:
29 Mar - 04 Apr 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
(cellular proliferation was not determined by incorporated radioactivity, thus no disintegrations per minute (DPM) and no SI values were calculated)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted Apr 2002
Deviations:
yes
Remarks:
(cellular proliferation was not determined by incorporated radioactivity, thus no disintegrations per minute (DPM) and no SI values were calculated)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
Landesanstalt für Pflanzenbau und Pflanzenschutz, Mainz, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approxiamtely 7 - 8 weeks
- Weight at study initiation: 17.0 - 20.6 g
- Housing: The animals were individually housed in Makrolon type I cages.
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 29 Mar 2005 To: 04 Apr 2005

Study design: in vivo (LLNA)

Vehicle:
other: acetone
Concentration:
3, 10 and 50% (w/w)
No. of animals per dose:
6
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Lymph node weight, cell count and ear weight were determined for the control and treatment animals.
- Criteria used to consider a positive response: In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous administration of several concentrations of the test material to the skin of the skin at the back of the ear is determined. The parameters used to characterize the response are lymph node cell count and to a certain extent weight. Because not only sensitization induction but also irritation of the ear skin by the test material may induce lymph node responses, the weight of ear punches taken from the area of test material application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test material.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test material was applied to the entire dorsal surface of each ear of each mouse on Days 1, 2 and 3 in concentrations of 3, 10 and 50% in acetone. The irritation effects on the treatment site were assessed daily.
Three days after the last application the animals were sacrificed. Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear. The weight of the pooled punches was determined for each animal. Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal. After weight determination, the pooled lymph nodes of each animal were stored in phosphate
buffered saline in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 6 mL of phosphate-buffered physiological saline. For determination of cell count the standard suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter. Each suspension was measured in triplicate (3 dilutions of the standard suspension). The mean of the triplicate measurements was used for further evaluation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The indices of Iymph node weight, cell count and ear weight were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group. Statistical analyses for lymph node weight, cell count and ear weight were performed with the Wilcoxon-Test.

Results and discussion

Positive control results:
Studies using the positive control substance alpha-hexylcinnamaldehyde, techn. 85% are performed twice a year in the Iaboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen. In a study performed from 26 Apr to 10 May 2005 at the same testing facility, 1% alpha-hexylcinnamaldehyde in acetone showed skin sensitising properties (project # 45H0288/982059). Lymph node weight indeces were 1.12, 1.37 (p ≤ 0.05) and 1.96 (p ≤ 0.01), the cell count indeces were 1.28 (p ≤ 0.05), 1.63 (p ≤ 0.05) and 2.94 (p ≤ 0.01) and the ear weight indeces were 1.04, 1.06 (p ≤ 0.05) and 1.14 (p ≤ 0.01) in the animals treated with 1, 3 and 10% alpha-hexylcinnamaldehyde in acetone, respectively.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Test group / Remarks:
3% application
Remarks on result:
not measured/tested
Parameter:
SI
Test group / Remarks:
10% application
Remarks on result:
not measured/tested
Parameter:
SI
Test group / Remarks:
50% application
Remarks on result:
not measured/tested
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The test material did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as 3% or 10% preparations in acetone. The minimal but statistically significant increase in mice treated with the 50% test substance preparation was too small to be considered biologically relevant. The statistically significant increases in ear weights indicate irritation of the ear skin at all concentrations. Thus, the minimal increase in cell count and lymph node weight index in test group 4 is considered to be related to ear skin irritation produced by the combination of vehicle and test substance, which starts already at the 3% concentration without relevant lymph node response. Higher concentrations were not tested, because the high concentration tested (50%) produced ear skin irritation.

CLINICAL OBSERVATIONS:
1/6 animals of the 50% dose group was found dead on study day 2. No macroscopic pathologic abnormalities were noted. The death was not considered to be test substance related.

BODY WEIGHTS
Body weights of the treatment and the control group were within the normal range.

Any other information on results incl. tables

Table 1 Results of LLNA

Test Group Treatment Lymph Node Weight [mg] Cell Count [Counts/Lymph Node Pair] Ear Weight [mg]
    Mean S.D. Index1 Mean S.D. Index1 Mean S.D. Index1
1 vehicle acetone 5.5 0.6 1.00 9,588,333 1,972,130 1.00 26.9 0.3 1.00
2 3% in acetone 5.5 0.6 1.00 9,998,333 1,167,985 1.04 28.0 0.4 1.04 ##
3 10% in acetone 5.7 1.0 1.04 11,212,333 2,641,081 1.17 28.9 0.8 1.07 ##
4 50% in acetone 7.7 1.1 1.41 ## 15,870,000 4,998,814 1.66 ## 29.9 1.4 1.11 ##

S.D. = standard deviation

* test group x / test group 1 (vehicle control)

# = statistically significant for the value p 0.05

## = statistically significant for the value p 0.01

Applicant's summary and conclusion

Interpretation of results:
other: non-sensitising according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: not classified