Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- modification: in addition, measurements of ear swelling and ear weight were done to discriminate the irritating potential from the sensitizing potential of the test substance (Integrated Model for the Differentiation of Skin reactions (IMDS))
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised 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
- Source: harlan Nederland, 5960 AD Horst, The Netherland
- Mean weight at study initiation: 26 - 36 g
- Housing: 8 animals per cage during adaption period; 1 animal per cage during the study
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2
- rel. Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 2, 10, 30 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the positive control or the vehicle were 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 25µl/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
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 draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
- body weights at the start and 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 the Mann-Whitney or the Wilcoxon signigicance test (Rank Sum Test or One Way ANOVA or Kruskal-Wallis ANOVA) at significance levels of 5 % (one-tailed for LLNA/IMDS or PNLA (larger)). 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

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: No significant dose-dependent increase in the stimulation indices for the weight or cell counts as well as for ear swelling or ear weights.

Any other information on results incl. tables

Table 1: Summary of the LLNA/IMDS results (means of 6 animals per group)

Parameter investigated

Vehicle

control

Dose 2 %

 Dose 10 %

Dose 30 %

Stimulation index:

weight of draining lymph nodes

1.00

0.89

1.25

1.11

Stimulation index:

cell count in draining lymph nodes

1.00

0.88

1.21

1.20

Ear swelling in 0.01 mm on day 4 (index)

18.25

(1.00)

18.42

(1.01)

18.25

(1.00)

18.08

(0.99)

Ear weight in mg / 8 mm diameter punch on day 4 (index)

14.01

(1.00)

13.84

(0.99)

13.63

(0.97)

13.34*

(0.95)

*statistically significant increase (p <= 0.05)

The test item has no sensitizing potential in mice after dermal application of up to and including a 30 % concentration. No indication for a non-specific (irritant) activation was detected either. Therefore, the concentration of 30 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization. These findings were verified by the results obtained with the positive control compound Alpha Hexyl Cinnamic Aldehyde.

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

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Executive summary:

The test substance was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 2, 10 and 30 % formulated in dimethylformamide were tested. The results show that the test item has neither a non-specific (irritant) nor a specific immunostimulating (sensitizing) potential.These findings were verified by the results obtained with the positive control.