Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 February - 6 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to EU method and in compliance with Good Laboratory Practice.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tropicate
- Substance type: pure active substance
- Physical state: liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
The animals were female (CBA/J@Rj) strain mice from Elevage Janvier, Le Genest-Saint-Isle, France. 8 weeks old animals, nulliparous and nonpregnant, were selected having body weights in the range of 18.7-21.6 g+/-20% of the sex mean.
A controlled environment was maintained in the room with optimal conditions of approximately 15/h air changes, a temperature of 22±3ºC, a relative humidity of 30-70% and a 12 hour artificial fluorescent light/12 hour dark cycle per day.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
Drinking water (tap) and food were supplied freely.
After an acclimatisation period of 5 days, animals were randomly selected and marked on the tail.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Three dose groups of four animals per group were treated for three consecutive days with the test item. The concentrations of the dose groups were 100% (undiluted), 50% (v/v), 25% (v/v) and 0% (negative control).
No. of animals per dose:
4 mice per dose group.
Details on study design:
RANGE FINDING TESTS:
The prweliminary test was ascreening of the daily clinical observation, the body weigth evolution measured on Day 1 and Day 6, the ear thickness measurement on Day 1, 3 and 6, the weight of the lymph nodes and the cell count.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Criteria used to consider a positive response: If the EC1.4 for any of the treated groups ≤100%, the test item is classified as a sensitiser. EC1.4 is the theoretical concentration resulting in a SI value of 1.4.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl of the test compound (and negative control) was daily applied to the entire dorsal surface of each ear of each mouse using the tip of a micro pipette for three consecutive days. On day 6, the mice were killed with an intraperitoneal injection of sodium pentobarbital. The draining Auricular lymph node were excised and a single cell suspension was prepared by pooling the lymph node cells of four mice through mechanical tissue disaggregation in 4ml of PBS containing 0.5% BSA into a well of a multi-well 6.
10µl of the cell suspension was diluted in physiological saline solution (NaCl 0.9%). The lymphocytes were counted using a Beckman Coulter Z2 cell counter. The lower and upper selected sizes were 5µm and 15µm resp.

The weight of the animals were recorded prior to exposure to the test compound and on day 6 prior to the intraperitoneal injection. The thickness of the right ear of the animals were measured using a micrometer prior to exposure on day 1, prior to the third exposure on day 3 and after the intraperitoneal injection. Punch biopsies of 8mm in diameter of the apical area of both ears were prepared and weighed on day 6 in order to assess the irritation potential.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC1.4 is determined by linear interpolation of the points in the dose-response curve immediately above and below the 1.4 threshold.

Results and discussion

Positive control results:
A study was performed to assess the sensitivity of the strain mouse with the known sensitiser (positive control) α-hexylcinnamaldehyde (95% pure, CAS 101-86-0). Three groups, each of four animals were treated with 50µl (25µl/ear) at concentrations of 5%, 10% and 25% as a solution in acetone/olive oil (4:1 v/v). A negative control group was included. The EC1.4 value of the positive control was 8.73%.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: The stimulation index (SI) calculated by pooled approach was 1.23, 1.72 and 2.16 for the treated groups with test substance concentrations of 25%, 50% and 100%.

Any other information on results incl. tables

Preliminary test

No significant clinical abnormalities or increase in ear thickness was noted in the animal treated at 100%. the body weight on day 1 and 6 were 18.8 and 18.1 g resp. Ear weight on day 6 was 26.0 mg. The weight of the lymph nodes was 6.7 mg and the cell count 14.85·106/ml. The test item is not considered as excessively irritant at the three concentrations. The concentration of 100% has therefore been choosen as the highest concentration for the main test.

Main test

No mortality and no signs of systematic toxicity were noted in test and control animals.

There were no signs of excessive irritation up to the concentration of 100%. On day 6, a slight dryness was noted on the treatment site of all animals treated at 100%.

Body weight evolution of the test animals between day 1-6 were comparable to the control group.

No significant ear thickness and ear weight were observed in the treated animals.

The cell count, stimulation index and calculation of EC1.4 can be found in the table below.

Group concentration Cell count
(x10^6 cells/ml)
Stimulation
Index
Result EC1.4
1 0% 24.06 n.a.
2 25% 29.66 1.23 Negative 33.67%
3 50% 41.43 1.72 Positive
4 100% 51.93 2.16 Positive

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The stimulation index is in excess of 1.4 and therefore test substance should be regarded as a skin sensitiser.
Executive summary:

The skin sensitisation potential of the test substance Tropicate was assessed according to OECD method 429, in the CBA/J strain mouse following topical application to the dorsal ear surface.

Three groups of four animals were treated for three consecutive days (day 1,2 and 3)with 50µl (25µ per ear) with the test compound in acetone/olive oil (4:1 v/v) at concentrations (v/v) of 100% (undiluted), 50%, 25% and 0% (negative control).

On day 6, he proliferation of the lymphocytes in the draining Auricular lymph nodes was determined by cell counting.

No mortality and no signs of systematic toxicity were found in all the animals at all concentrations. A slight dryness of was noted in the on the treatment site of al animals treated at 25%, 50% and 100%. No significant thickness in ear thickness and ear weight were observed at all concentrations. The test substance was therefore not considered as excessively irritant.

The stimulation index (SI) calculated by pooled approach was 1.23, 1.72 and 2.16 for the treated groups with test compound concentrations of 25%, 50% and 100%. The EC1.4 is 33.67% determined by linear interpolation. Since the SI>1.4 at concentrations of 50% and 100% (or EC1.4≤100%), the test substance is classified as a skin sensitiser.