2H-1-Benzopyran-2-methanol, alpha,alpha'-[[(phenylmethyl)imino]bis(methylene)]bis[6-fluoro-3,4-dihydro-, (alphaR,alpha'R,2R,2'S)-rel-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-07-20 to 2005-08-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2H-1-Benzopyran-2-methanol, alpha,alpha'-[[(phenylmethyl)imino]bis(methylene)]bis[6-fluoro-3,4-dihydro-, (alphaR,alpha'R,2R,2'S)-rel-

EC Number:

618-043-4

Cas Number:

876666-07-8

Molecular formula:

C29H31F2NO4

IUPAC Name:

2H-1-Benzopyran-2-methanol, alpha,alpha'-[[(phenylmethyl)imino]bis(methylene)]bis[6-fluoro-3,4-dihydro-, (alphaR,alpha'R,2R,2'S)-rel-

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-17303806-AAA (T001586)
- Physical state: solid, powder
- Appearance: white, beige

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT001586G1B121
- Expiration date of the lot/batch: 31-12-2005
- 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:
0.01 g/L in water
6.9 g/L in ethanol
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (DMSO) 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.

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: 6-8 weeks at the beginning of acclimation
- Weight at study initiation: 16.0 - 19.3 g
- Housing: individually in Makrolon Type I cages with wire mesh tops and granulated soft wood bedding
- Diet: ad libitum, pelleted standard diet
- Water: ad libitum, tap water
- Acclimation period: under test conditions after health examination (duration not specified). Only animals without any visible signs of illness were uesd for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3°C
- Humidity (%): 30-92%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 4, 8 and 16% (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 0.01 g/L in water, 6.9 g/L in ethanol
- Irritation: In a pre-test in two mice, test substance concentrations of 2, 4, 8 and 16 % (w/v) were tested; the treated animals did not show any signs of toxicity or irritation.
- Lymph node proliferation response: not measured (only determine highest non-irritant and technically applicable test item concentration)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Determination of incorporated 3HTdR
- Criteria used to consider a positive response: A test substance 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; and
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 was quantitatively added. The test substance concentrations were prepared serially. Homogeneity of the test substance in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion.
- 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. The application volume, 25 uL, was spread over the entire dorsal surface (diameter of circa 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of DMSO. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test substance applied.
- Five days after the first topical application, all mice were administered with 250 µL of 81.3 µCi/mL 3HTdR (corresponds to 20.325 µCi 3HTdR per mouse) by intravenous injection via a tail vein.
- Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of Na-thiopental.
The draining lymph nodes were rapidly excised and pooled for each experimental group (2 nodes per animal). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately 4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of Ultima Gold scintillation liquid and thoroughly mixed.
- The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1-mL-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

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 substance required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the stimulation index value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:

- Positive control study performed August 2004.
- The stimulation indices of the positive control groups were 1.06, 1.81 and 4.00 for the 5, 10 and 25% concentrations, respectively.
- The corresponding EC3 value was 18.2% (w/v).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.39

Test group / Remarks:

4% w/v group

Results 2

Parameter:

SI

Value:

1.26

Test group / Remarks:

8% w/v group

Results 3

Parameter:

SI

Value:

2.23

Test group / Remarks:

16% w/v group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DPM per lymph node (8 lymph nodes in total):
4% w/v group: 1061.9
8% w/v group: 960.3
16% w/v group: 1704.2

DETAILS ON STIMULATION INDEX CALCULATION
see Results table

EC3 CALCULATION
The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

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

BODY WEIGHTS
The body weight of the animals, recorded prior to the 1st application and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance T001586 was not a skin sensitizer under the described conditions in a mouse local lymph node assay and should therefore not be classified (S.I. values <3 for all tested concentrations).