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:
2005
Report Date:
2005

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 lymphnode 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 2 hours.

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)
- Sex: female
- Source: Harlan Winkelmann GmbH, 33176 Borchen, Germany
- Age at study initiation: 10 weeks
- Weight at study initiation: 28-33 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 7 days


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

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
vehicle, 2%, 10% or 50%
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The formulated test item was applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days. The volume administered was 25µl/ear. The concentrations were based on the experiences with the test system and the properties of the test substance.

EXAMINATIONS:
Autopsies - The animals were anaesthetized by inhalation of CO2 and sacrificed one day after the last application. The appropriate organs were then removed. Lymphatic organs were transferred into sterile physiologic saline solution.
Weight and cell count determinations - The weights of the lymph nodes were determined. . The cell counts per ml were determined. The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is always about 1.00 (+/- standard deviation), and the indices of vehicle treated animals are set to 1.00 (+/- standard deviation).
Ear Swelling - On day 0 and day 3 of the study the ear swelling of the animals was measured, and mean ear swelling was calculated.
Ear weight - On day 4 of the study the ear weight of the sacrificed animals was measured using a punch to take of a piece of every ear with a diameter of 8 mm.
Body weights - The body weights of the animals were recorded initiating the study and at the end of the 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 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:
A recently conducted local lymph node assay with alpha hexyl cinnamic aldehyde showed a clear sensitising potential.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Cell counts were dose-dependently increased in all dose groups. 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 %) / 3.58 (2 %) / 3.82 (10 %) / 4.69 (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 was a clear dose dependent increase in weights of the draining lymph nodes:

weight index 1.00 (0%) / 2.55 (2%) / 2.92 (10%) / 3.59 (50%).

The "positive level" of ear swelling which is 2 x 10exp-2 mm increase, i.e. about 10% of the control values, has been exceeded in all dose groups:

ear swelling day 1 = 17.42 (0 %) / 17.67 (2 %) / 17.25 (10 %) / 17.08 (50 %); day 4 = 16.83 (0 %) / 20.33 (2 %) / 22.42 (10 %) / 25.25 (50 %); Index day 4 = 1.00 (0 %) / 1.21 (2 %) / 1.33 (10 %) / 1.50 (50 %).

A significant increase compared to vehicle treated animals regarding ear weights was also detected in all dose groups: ear weight day 4 = 10.79 (0 %) / 12.92 (2 %) / 14.05 (10 %) / 17.98 (50 %); Index day 4 = 1.00 (0 %) / 1.20 (2 %) / 1.30 (10 %) / 1.67 (50 %).

An increase in ear swelling and ear weights would point to an acute irritating (inflammatory) response. However, such an irritating property is also combined with a strong skin sensitizing potential of a test compound.

Applicant's summary and conclusion

Executive summary:

A modified Local Lymph Node Assay (IMDS; OECD TG 429) was performed on 6 female NMRI mice/group with epicutaneously applied test substance concentrations of 0% (vehicle control), 2%, 10% and 50%. Compared to vehicle treated animals there was a clear increase in weights of the draining lymph nodes and in the cell counts at all dose groups. Additionally an increase in ear swelling and ear weight was detected; this would point to an acute (inflammatory response). However, such an irritating property can also be combined with a strong sensitising potential of the test substance.