Bicyclo[2.2.1]heptane-1-methanesulfonic acid, 7,7-dimethyl-2-oxo-, (1R,4S)-, compd. with rel-(.alpha.R,.beta.S)-.alpha.-(2,4-difluorophenyl)-5-fluoro-.beta.-methyl-.alpha.-(1H-1,2,4-triazol-1-ylmethyl)-4-pyrimidineethanol (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 June to 2 July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

188416- 34-4

IUPAC Name:

188416- 34-4

Constituent 2

Reference substance name:

(2RS,3SR)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butanol (1R)- 10- camphorsulphonate

IUPAC Name:

(2RS,3SR)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butanol (1R)- 10- camphorsulphonate

Details on test material:

- Analytical purity: 94.0%
- Lot/batch No.: 03207003

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: 8-12 weeks (beginning of acclimatization period)
- Weight at study initiation: 14.5-16.4 g (beginning of acclimatization period)
- Housing: Housed individually in Makrolon type-2 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no.84/02 mouse maintenance diet available ad libitum. Results of analyses for contaminants are archived at RCC.
- Water (e.g. ad libitum): Community tap water from Itingen, available ad libitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3℃
- Humidity (%): relative humidity 30-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light /12 hour dark cycle with at least 8 hours music during the light period.

Study design: in vivo (LLNA)

Vehicle:

other: Ethanol: water, 7:3 (v/v)

Concentration:

The test item at concentrations of 5%, 10% and 25% (w/v) in ethanol: water, 7:3(v/v)

No. of animals per dose:

Number of animals for the pre-test (non-GLP): 2 females
Number of animals for the main study: 20 females (reserve animals will be used as needed and listed in raw data and report)
Number of animals per group: 5 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of negative control: 1

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Test system: Mice, CBA/CaOlaHsd
- Criteria used to consider a positive response: --First, that exposure to at least one concentration of the test item resulted in an incorporation of HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).
--Second, that 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 item was placed into a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added. The weight dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
1) Topical application
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 (v/v). The application volume, 25 μl, was spread over the entire dorsal surface (ø~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.
2) Administration of H-Methyl Thymidine
H-methyl thymidine was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 μl of 83.50 μCi/ml HTdR (equal to 20.9 μCi HTdR) by intravenous injection via a tail vein.
3) Determination of incorporated HTDR
Approximately five hours after treatment with HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL.
The draining lymph node cells were rapidly excised and pooled for each individual animal (2 nodes per mouse). 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 ℃ for at least 18 hours for precipitation of precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background HTdR level were also measured in two 1ml-aliquots of 5% trichloroacetic acid. The ß-scintillation counter expresses HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde

Statistics:

The mean values and standard deviations of body weights and DPM/mouse value were calculated.

Results and discussion

Positive control results:

In this study STIMULATION INDICES of 2.5, 3.7 and 9.7 were determined with the test item at concentrations of 5%, 10% and 25% (w/v) in acetone:olive oil, 4:1 (v/v).
A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).
The test item Alpha-Hexylcinnamaldehyde was found to be a sensitizer and an EC2 value of 7.08% (w/v) was derived.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

For Control group, there is no S.I. (SD); For test group at concentrations of 5% (w/v) in ethanol:water, 7:3 (v/v), the S.I. (SD) is 1.0 (0.3); For test group at concentrations of 10% (w/v) in ethanol:water,7:3 (v/v), the S.I. (SD) is 1.3 (0.1); For test group at concentrations of 25% (w/v)in ethanol:water,7:3 (v/v), the S.I. (SD) is 1.4 (0.4).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The level of HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background HTdR levels were also measured in two 1ml-aliquots of 5% trichloroacetic acid. The ß-scintillation counter expresses HTdR incorporation as the number of radioactive disintegrations per minute(DPM). For Control group, the DPM is 681±180; For test group at concentrations of 5% (w/v) in ethanol:water, 7:3 (v/v), the DPM is 681±180; For test group at concentrations of 10% (w/v) in ethanol:water, 7:3 (v/v), the DPM: 894±87; For test group at concentrations of 25% (w/v) in ethanol:water, 7:3 (v/v), the DPM: 964±267.

Any other information on results incl. tables

Mortality: No deaths occurred during the study period.

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

Body weights: Within the range commonly recorded for animals of this strain and age.

Group	% (w/v)	dpm/Mouse M±SD	S.I. (SD)	Statistical Analysis Dunnett-test (G=4, N=20, t=2.59)
				t Value	Conclusion
CG 1	---	681±180	--	--	--
TG 2	5	697±187	1.0 (0.3)	0.13	--
TG 3	10	894±87	1.3 (0.1)	1.77	--
TG 4	25	964±267	1.4 (0.4)	2.35	--

--no significant different at p≤0.05 two sides

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

In this study stimulation indices of 1.0, 1.3 and 1.4 were determined with the test item at concentrations of 5%, 10% and 25% (w/v) in ethanol:water, 7:3 (v/v).
A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).
Based on these criteria, the test item was found to be a non-sensitizer when tested at up to the highest achievable concentration of 25% (w/v) in ethanol:water, 7:3 (v/v).
Dunnett-test
No signification difference of dpm/mouse was determined at up to the highest achievable concentration of 25% (w/v) comparing with the vehicle control group at p≤0.05 (two sides).