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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
08 Nov 2011 - 30 Jan 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 429). For justification of read-across see CSR chapter 1.3.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Reason for selection of species: this inbred strain was chosen on the basis of previous studies performed in our laboratory, in which CBA/J mice showed the best proliferative response. Females have been chosen since this sex is recommended by regulatory authorities for this type of study.
Breeder: Janvier, Le Genest-Saint-Isle, France.
Age/weight: on the beginning of the treatment period, the animals of the preliminary test were approximately 11 to 12 weeks old and the animals of the main test were approximately 8 weeks old. In the main test, they had a mean body weight of 21.5 g (range: 19.5 g to 23.2 g).
Receipt: upon arrival at CIT, the animals were given a clinical examination to ensure that they are in good condition.
Allocation to groups: upon arrival at CIT, the animals were allocated to the groups (by sex) using a manual randomization procedure. A larger number of animals than necessary was acclimated to permit the selection and/or replacement of individuals.
Acclimation: the animals were acclimated to the study conditions from 13 days before the beginning of the treatment period. At the end of acclimation period, the required number of animals were selected according to clinical condition and body weight.
Identification: the animals were individually identified on the tail using an indelible marker (unique CIT identity number).

Environmental conditions
From arrival at CIT, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
. temperature : 22 ± 2°C,
. relative humidity : 50 ± 20%,
. light/dark cycle : 12 h/12 h (7:00 - 19:00),
. ventilation : approximately 12 cycles/hour of filtered, non-recycled air.
The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are recorded continuously and the records checked daily and filed.
Housing
The animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages (Techniplast 1145T, 435 cm2, 36.9 x 15.6 x 13.2 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Each cage contained two enrichments (mouse hut and cocoon). In the main test, on day 6 before the 3H-TdR injections, the animals were individually housed in disposable crystal polystyrene cages (22.0 cm x 8.5 cm x 8.0 cm).
Food and water
All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batches No. 9615507 and 7055973 (SSNIFF Spezialdiäten GmbH, Soest, Germany) and tap water (filtered using a 0.22 micron filter) contained in bottles.
Contaminant analyses
The batches of diet and sawdust were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants that could have interfered with or prejudiced the outcome of the study were found in the diet, drinking water or sawdust.

Study design: in vivo (LLNA)

Vehicle:
other: 1% pluronic PL 92 in purified water treated by reverse osmosis
Concentration:
0, 10, 25, 50%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A homogeneous suspension was obtained at the concentration of 50% in 1% pluronic PL 92.
- Irritation:
To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was performed in four animals.
The treatment groups for the preliminary test are detailed in the following table:
Group Number of animals Concentration (%) Left ear - Right ear
1 2 females 5 10
2 2 females 25 50
The irritation level of the test item was determined according to the following table:
% increase in ear thickness
between day 1 and day 6 Irritation level Interpretation
< 10% I Non-irritant
10 - 25% II Slightly irritant
≥ 25% III Irritant

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: AURICULAR LYMPH NODE CELL (ALNC) PROLIFERATION ASSAY:
Lymph node cell proliferative response was measured as described by Kimber and Dearman (1). On day 6 of the main test, all animals of all groups received a single intravenous injection of 250 μL of 0.9% NaCl containing 20 μCi of 3H-TdR (specific activity of 20 Ci/mmol) via the tail vein.
Approximately 5 hours later, the main test animals were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of ALNC was prepared by mechanical disaggregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed once with 15 mL of 0.9% NaCl. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic (TCA) at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three millilitres of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting.
The results were expressed as disintegrations per minute (dpm) per group and as dpm/node.
Stimulation Indices (SI) were calculated according to the following formula: SI = dpm per node of the treated group / dpm per node of the vehicle control group.
- Criteria used to consider a positive response:
The test item is considered as a skin sensitizer when the SI for a dose group is ≥ 3 together with consideration of a dose-response relationship. Other relevant criteria such as radioactivity levels and ear thickness are also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was administered as a homogeneous suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed under magnetic agitation with the required quantity of vehicle. The test item concentrations were expressed in % (w/v). The positive control was prepared at the concentration of 25% (v/v) a mixture of Acetone/Olive Oil (4/1; v/v) (AOO). Dosage forms preparations were prepared by the CIT Pharmacy extemporaneously on the day of each administration. The dosage forms were stored at room temperature and delivered to the study room in brown flasks.
Reagent used for the proliferation assay
The reagent used for the proliferation assay was [3H] methyl-thymidine (3H-TdR), batch No. 201111, supplied by PerkinElmer (Courtaboeuf, France).
Three days before the injections, the required quantity of 3H-TdR was diluted in 0.9% NaCl (20 μCi in 250 μL of 0.9% NaCl per animal). The obtained solution was stored at +4°C and protected from light before use.
On days 1, 2 and 3, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group. The experiment was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Doses: Vehicle Test item 10%, 25%, 50%; HCA 25% Stimulation index (SI): -, 0.40, 0.64, 0.82, 5.02
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Doses: Vehicle Test item 10%, 25%, 50%; HCA 25% dpm per group: 2434, 971, 1546, 1990, 12229

Any other information on results incl. tables

Table 1. Proliferation assay (mean values)

Groups

Treatment and conditions


Number of nodes per group

dpm per group

dpm per node

Stimulation index (SI)

Increase in ear thickness (% between day 1 and day 6)

Irritation level

EC3 value

3

Vehicle

8

2434

304.25

 

0.00

 

NA

4

Test item 10%

8

971

121.38

0.40

-6.80

I

5

Test item 25%

8

1546

193.25

0.64

-1.98

I

6

Test item 50%

8

1990

248.75

0.82

-6.06

I

7

HCA 25%

8

12229

1528.63

5.02

 

 

NA = not applicable

dpm = disintegrations per minute

I = non-irritant (increase in ear thickness < 10%)

II = slightly irritant (increase in ear thickness = 10 to 25%)

III = irritant (increase in ear thickness > 25%)

EC3 value = theoretical concentration resulting in a SI value of 3

stimulation index = dpm of treated group / dpm of control group

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions of this study, the test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). A preliminary test was performed to define the test item concentrations for the main test. Two groups of two female mice received the test item by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 5, 10, 25 or 50% under a dose-volume of 25 μL. The thickness of both ears of each animal was measured and the local reactions were recorded. In the main test, three groups of four female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 10, 25 or 50% under a dose-volume of 25 μL. Negative (vehicle) and positive controls (α-hexylcinnamaldehyde (HCA)) were conducted. From day 1 to day 3 and on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-TdR. The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

No unscheduled deaths and no clinical signs were observed in any animals. Body weight was unaffected by the test item-treatment. Alopecia was observed on day 6 in 3/4 females treated at 50%. No notable increase in ear thickness was observed at any tested concentration. The threshold positive value of 3 for the SI was reached in the positive control group. The experiment was therefore considered valid. No notable lymphoproliferation was noted with the test item at any tested concentration. Therefore the test subtance was not a skin sensitizer under the conditions of this study.