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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
Study period:
from September 14th to 26th, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
Adopted July 22nd 2010.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:JN
Sex:
female
Details on test animals and environmental conditions:
- Age: 7 to 8 weeks old
- Supplier: Charles River Italia S.p.A., Calco (Lecco), Italy
- Breeder: Charles River France Laboratories, Domaine des Oncins B.P.0109, F 69592 L’ARBRESLE CEDEX, France
- Weight range at arrival: 16 to 21 grams
- Acclimatisation period: at least 5 days
- Veterinary health check: during acclimatisation period
- Animals per cage: up to 5 during acclimatisation; 1/cage during the study
- Housing: Polysulphone solid bottomed cages measuring 35.5 × 23.5 × 19 cm with nesting material
- Cage control: daily inspected and changed as necessary (at least twice/week)
- Water: drinking water supplied to each cage via a water bottle; Ad libitum
- Diet: 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
- Diet supply: Ad libitum throughout the study
- Room lighting: artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
- Air changes: approximately 15 to 20 air changes per hour
- Temperature range: 22 °C ± 2 °C
- Relative humidity range: 55 % ± 15 %

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 10 and 5 % (w/w)
No. of animals per dose:
13-19 (vehicle group), 21-27 (5% w/w group), 29-35 (10 % w/w group), 37-43 (25 % w/w group), 45-31 (positive control group)
Details on study design:
PRELIMINARY TEST
- Concentrations tested: 50, 25, 10, 5 and 2.5 % (w/w).
- Frequency of treatment: the animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle or test item formulations, depending on the animal.
- Dosing method: a dose volume of 25 µl/ear/day of the appropriate concentration was applied to the dorsal surface of each ear (50 µl/animal/day), using a micropipette.
- Observations: throughout the study, all animals were checked twice daily for mortality and morbidity. The animals were observed for clinical signs on: Day 1 before and 1 hour after dosing and on day 2 to 6: daily (approximately 1 hour after daily dosing, when applicable). The animals were weighed at allocation (Day 1) and on sacrifice (Day 6).
- Scoring of dermal reaction: the treated sites of all animals were examined daily (once before first dosing, before dosing on Days 2 and 3 and daily thereafter) for erythema and eschar formation.
- Ear thickness measurement: the ear thickness was measured by a suitable micrometer on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6.
- Termination: the animals were sacrificed on Day 6 by carbon dioxide narcosis. After sacrifice, regularly shaped biopsies were obtained from both ears (ear punch) and weighed together. No necropsy was performed on the animals.

SELECTION OF DOSE LEVELS: in the main assay, the test item was used at the higher concentrations that were systemically tolerated and judged not to cause excessive local skin irritation. In particular, concentrations are considered irritant if:
– erythema grading (score) is ≥ 3 at any day of measurement and/or
– ear thickness is ≥ 25 % with respect to Day 1 and/or
– ear punch weight is ≥ 25 % with reference to the negative control group
No signs of toxicity (significant clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations in the preliminary test. The evaluation of visible reactions showed no erythema at any of the concentrations investigated (50, 25, 10, 5 and 2.5 %w/w).
The evaluation of ear punch weight indicated a relevant increase only at the highest concentration tested (50 % w/w). Although no evident alterations were seen in the other two parameters (evaluation of erythema and ear thickness), the highest concentration selected for the Main Assay was 25 % w/w.

MAIN STUDY:
- Dosing: the animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle, test or positive control item formulations. The animals were treated intraperitoneally, on Day 5 (once only), with 0.5 ml/animal of a solution of BrdU at a concentration of 10 mg/ml in physiological saline (0.9 % NaCl), using a plastic graded syringe.
- Dosing method: a dose volume of 25 µl/ear/day of each selected concentration and controls was applied to the dorsal surface of each ear (50 µl/animal/day), using a micropipette
- In vivo observations: throughout the study, all animals were checked twice daily for mortality and morbidity. The animals were observed for clinical signs on: Day 1: before and 1 hour after dosing and on Day 2 to 6: daily (approximately 1 hour after daily dosing, when applicable. The animals were weighed at allocation (Day 1) and on sacrifice (Day 6).
- Euthanasia: the animals were killed on Day 6, approximately 24 hours after BrdU injection, by carbon dioxide narcosis. No necropsy was performed on the sacrificed animals.
- Lymph node collection: shortly after sacrifice of each animal, the auricular lymph nodes were rapidly excised, pooled on individual basis and individually collected in a solution of 2 % BSA-PBS [2 % bovine serum albumine (BSA) in phosphate buffered saline, PBS]. Cell suspensions were prepared for the evaluation of proliferation at lymph node.

DETERMINATION OF CELLULAR PROLIFERATION
- Preparation of single cell suspension: a single cell suspension of lymph node cells (LNC) was prepared from each animal by gentle mechanical disaggregation and passage through a 70 µmnylon mesh. The suspensions thus obtained were centrifuged and each supernatant resuspended in 20 ml of 2 % BSA-PBS.
- Measurement of BrdU content in lymphocytes DNA: BrdU was measured by ELISA using a commercial kit (Roche Applied Science,Mannheim, Germany, Catalogue No. 11 647 229 001, batch No. 11417600), according to manufacturer instructions. Briefly, 100 µl of the LNC suspension were added to the wells of a flat-bottom 96-well microplate in triplicate. Two replicate microplates were prepared. After fixation and denaturation of the LNC, 100 µl of anti-BrdU antibody labelled with peroxidase were added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and 100 µl of the substrate solution were then added and allowed to produce chromogen. The reaction was finally stopped by adding 25 µl of stop solution (1M H2SO4). Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm). The replicate plate was destroyed after the evaluation of the results obtained in the first plate, since the objective of the study had been achieved.

CALCULATION OF BrdU LABELLING INDEX: the BrdU labelling index was calculated for each mouse and a group mean was subsequently calculated. Results for each treatment group were expressed as the mean Stimulation Index (SI). The SI was derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control groups by the mean labelling indices for the respective vehicle group.

CRITERIA USED TO INTERPRET A POSITIVE RESPONSE: the test item is considered to induce sensitisation when the SI for any single treatment dose group is ≥ 1.6. It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).
Positive control substance(s):
other: 25% (w/w) alpha-hexylcinnamaldehyde (Sigma Cat. W256900, batch no. MKBS3936V) in acetone:olive oil 4:1 v/v.
Statistics:
Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous, a Modified t test (Cochran and Cox) was applied.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.31
Test group / Remarks:
5 % w/w
Parameter:
SI
Value:
1.88
Test group / Remarks:
10 % w/w
Parameter:
SI
Value:
2.05
Test group / Remarks:
25 % w/w
Cellular proliferation data / Observations:
POSITIVE CONTROL RESULTS: a Stimulation Index of 4.38 was calculated. As it was greater than 2, the test was regarded as valid.

CLINICAL OBSERVATIONS: neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated [25, 10 and 5 % (w/w)].

BODY WEIGHTS: changes in body weight observed during the study were within the expected range for this strain and age of animals.

STATISTICS: Dunnett’s test showed a statistically significant difference (p < 0.05) between mid- and high dose groups, when compared to the negative control group.

Applicant's summary and conclusion

Interpretation of results:
other: classified as a Skin Sensitiser Cat.1, according to the CLP Regulation (EC) No.1272/2008
Conclusions:
The substance is a skin sensitiser as the SI is higher than 1.6 in the mid and high dose group.
Executive summary:

The ability of the test item to induce skin sensitisation in the CBA/JN mouse, using the Local Lymph Node Assay (LLNA: BrdU-ELISA method) according to the OECD Guideline 442b.

In the main assay, the test item was topically administered at the concentrations of 25, 10 and 5 % (w/w), in acetone:olive oil 4:1 (v/v) for three consecutive days. After one day of no treatment, BrdU solution was injected inter-peritoneally. Twenty four hours after BrdU injection, the animals were killed and the auricular lymph nodes were rapidly excised, pooled on individual basis and individually collected. Cell suspensions were prepared for the evaluation of proliferation at lymph node.

An increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups with SI of 2.05, 1.88 and 1.31 respectively, indicating that the test item may elicit a sensitisation response. Dunnett’s test showed a statistically significant difference between mid- and high dose groups, when compared to the negative control group.

The substance is considered to be a skin sensitiser as the SI is higher than 1.6 in the mid and high dose group.