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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:
2005-05-20 to 2005-08-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
1) The relative humidity higher than recommended; 2) The vehicle used was not listed in the OECD Guideline as a preferred vehicle. 3) animals may have been younger than recommended (age at end of acclimatization not clear)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-117676-AAA (T000263)
- Physical state: solid (powder)
- Appearance: White, slight beige amorphous, crystalline, micropowder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Test item delivered by the Sponsor Janssen Pharmaceutica N.V. with batch number: Code 8693
- Expiration date of the lot/batch: 31 December 2005
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle:
solubility: 0.34 g/L in water, > 340 g/L in ethanol
stability in solvent not indicated by the Sponsor
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was placed into a volumetric glass beaker on a tared balance and the vehicle (Ethanol:Water (7:3)) was quantitatively added. The test item concentrations were prepared serially.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 19.1 - 21.7 g, mean 20.2 +/- 0.8 g
- Housing: single cage type: Makrolon Type I, with wire mesh top. Bedding: granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3
- Humidity (%): 30 - 87, for the humidity there was a deviation from the study plan (upper limit), but it didn't affect the validity of the study
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: ethanol:water (7:3)
Concentration:
Pre-Experiment: 2.5, 5, 10, and 25% (w/v)
Main Experiment: 5, 10, and 25% (w/v)
No. of animals per dose:
2 females for pre-test
4 females (non-pregnant and nulliparous) in each test group.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 0.34 g/L in water and >340 g/L in ethanol
- Irritation: No signs of toxicity or irritation.
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
1) exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
2) the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- The test substance was placed into a volumetric flask glass beaker on a tared balance and the vehicle (Ethanol:Water (7:3)) was quantitatively added. The test item concentrations were prepared serially. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.

The preparations were made freshly before each dosing occasion.

To determine the highest non-irritant and technically applicable test item concentration, a pretest was performed in two mice with concentrations of 2.5, 5, 10 and 25% (wlv). At these concentrations, the treated animals did not show any signs of toxicity or irritation.

In the main experiment, concentrations of 5, 10, and 25 % were assayed.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10 and 25% (w/v) in Ethanol:Water (7:3). The application volume, 25 uL, was spread over the entire dorsal surface (diameter of ~8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

Five days after the first topical application, all mice were administered with 250 µL of 78.2 µCi/mL 3HTdR (corresponds to 19.55 µCi 3HTdR per mouse) by intravenous injection via a tail vein.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation:
EC3 = (a-c) [(3-d) / (b-d)] + c,
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the coordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
Test Group 2 (5% (w/v)): S.I. = 2.29
Test Group 3 (10% (w/v)): S.I. = 3.21
Test Group 4 (25% (w/v)): S.I. = 8.44
EC3=8.9% (w/v)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
4.52
Test group / Remarks:
5% w/v group
Parameter:
SI
Value:
9.99
Test group / Remarks:
10 % w/v group
Parameter:
SI
Value:
12.83
Test group / Remarks:
25 % w/v group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
5% w/v group: DPM: 10572.5 (8 lymph nodes)
10% w/v group: DPM: 23363.5 (8 lymph nodes)
25% w/v group: DPM: 30002.9 (8 lymph nodes)

DETAILS ON STIMULATION INDEX CALCULATION
In this study Stimulation Indices of 4.52, 9.99, 12.83 were determined with the test item at concentrations of 5, 10 and 25% (w/v) in Ethanol:Water (7:3).

EC3 CALCULATION
The EC3 value could not be calculated, since all SI's were above 3.

VIABILITY/MORTALITY
No deaths occurred during the study period

CLINICAL OBSERVATIONS:
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test substance T000263 was found to be a skin sensitizer under the described conditions.