2-[(5A,7A)-17-(CYCLOPROPYLMETHYL)-3,6-DIMETHOXY-18,19-DIHYDRO-4,5-EPOXY-6,14-ETHENOMORPHINAN-7-YL]-3,3-DIMETHYLBUTAN-2-OL

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2006-10-24 to 2006-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-[(1S,2R,6S,14R,15R,16R)-3-(cyclopropylmethyl)-11,15-dimethoxy-13-oxa-3-azahexacyclo[13.2.2.1²,⁸.0¹,⁶.0⁶,¹⁴.0⁷,¹²]icosa-7,9,11-trien-16-yl]-3,3-dimethylbutan-2-ol

Cas Number:

16524-65-5

Molecular formula:

C30H43NO4

IUPAC Name:

(2S)-2-[(1S,2R,6S,14R,15R,16R)-3-(cyclopropylmethyl)-11,15-dimethoxy-13-oxa-3-azahexacyclo[13.2.2.1²,⁸.0¹,⁶.0⁶,¹⁴.0⁷,¹²]icosa-7,9,11-trien-16-yl]-3,3-dimethylbutan-2-ol

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-18163678-AAA (T002713)
- Physical state: solid (powder)
- Appearance: White powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00477910 RT002713G4A051
- Expiration date of the lot/batch: December 03, 2006 (retest date)
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data
- Solubility of the test substance in the solvent/vehicle:
Solubility in water: < 10 g/l
Solubility in vehicle: Ethanol: 22.3 g/l; 10 % acetic acid: 170.5 g/l; 5 % phosphoric
acid: 96.5 g/l
No data available on stability of the test substance in the vehicle

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 (acetone:olive oil (4+1)) 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: females (nulliparous and non-pregnant), Harlan Netherlands, BV. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at beginning acclimatisation: 7 - 8 weeks
- Weight at study initiation: 17.6 +/- 0.9 g
- Housing: single, Makrolon Type I, with wire mesh top (EHRET GmbH, 0-79302 Emmendingen), granulated soft wood bedding (Harlan Winkelmann GmbH, 0-33178 Borchen)
- Diet: 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 (°C): 22 ± 3°C
- Humidity (%): 30 - 73%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

first main experiment: 6.25, 12.5, 25 % (w/v)
second main experiment: 12.5 % (w/v)

No. of animals per dose:

pretest: 2
main study: 4 per dose group (two experiments, in total 24 females)

Details on study design:

Test item preparation:
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (acetone:olive oil (4+1)) 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.

RANGE FINDING TESTS:

To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest was performed in two mice (pretest excluded from Statement of Compliance). The pretest data showed that the test item could be dissolved in acetone:olive oil (4+1) up to 25 % (w/v). The treatment of mice with 3.13, 6.25, 12.5, and 25 % test item in Acetone:olive oil (4+1) did not show any signs of severe local irritation or systemic toxicity.

MAIN STUDY

- Animal assignment:
Four animals were assigned to each group: first experiment: No. 1 control group, No. 2 (low dose group (6.25% test item concentration), No. 3 (mid dose group (12.5% test item concentration), No. 4 (high dose group (25% test item concentration)
In order to dermine the missing dose dependency of the response in the high dose group due to a possible false positive result at the mid dose a second experiment was performed with the mid dose (12.5%)
second experiment: No. 1 control group, No. 2 (mid dose group (12.5% test item concentration)

- 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 6.25, 12.5 and 25 % (w/v) in acetone:olive oil, 4+1 (v/v) in the first experiment and 12.5% in the second experiment. The application volume, 25 uI, was spread over the entire dorsal surface (average ca. 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).

-Administration of 3H-Methyl Thymidine:
Five days after the first topical application, all mice were administered with 250 uI of 79.4 uCi/ml /(first experiment), 80.4 uCi/ml /(second experiment) (corresponds to 19.85 uCi (first experiment), 20.1 uCi (second experiment) 3HTdR per mouse) by intravenous injection via a tail vein.

-Determination of Incorporated 3HTdR:
Approximately five hours after intravenous injection, the mice were euthanised, the draining auricular lymph nodes excised and pooled per group (8 nodes per group). Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid for at least 18 hours. The precipitates were then resuspended in 5% trichloroacetic acid (1 ml) and transferred to glass 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. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of Raw Data:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

- Criteria used to consider a positive response:
-First, that 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.
- 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.
The decision to select a STIMULATION INDEX (S.I.) of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated for body weights.
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 co-ordinates of the two pair 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 5% test item conc., S.I: 2.04
Test group 10% test item conc., S.I: 6.31
Test group 25% test item conc., S.I: 12.45
An EC3 of 6.1% (w/v) was calculated.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.21

Test group / Remarks:

6.25 % w/v group (experiment 1) - based on results of 4 animals

Results 2

Parameter:

SI

Value:

4.01

Test group / Remarks:

12.5 w/v% group (experiment ) - based on results of animals

Results 3

Parameter:

SI

Value:

5.89

Test group / Remarks:

12.5 % w/v group (experiment 2) - based on results of 4 animals

Results 4

Parameter:

SI

Value:

2.51

Test group / Remarks:

25% w/v group (experiment 1) - based on results of 4 animals

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
1st main experiment:
control group: 699.6 DPM per lymph node (8 lymph nodes in total)
6.25 % w/v group: 1542.8 DPM per lymph node (8 lymph nodes in total)
12.5% w/v group: 2803.2 DPM per lymph node (8 lymph nodes in total)
25% w/v group: 1756.9 DPM per lymp node (8 lymph nodes in total)
2nd main experiment:
control group: 488.5 DPM per lymp node (8 lymph nodes in total)
12.5% w/v group: 2877.7 DPM per lymp node (8 lymph nodes in total)

DETAILS ON STIMULATION INDEX CALCULATION see Results table

EC3 CALCULATION
Test item concentration % S.I.
Group 1 6.25 (a) 2.21 (b)
Group 2 12.5 (c) 4.01 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 9% (w/v)
EC3 = Estimated concentration for a S.I. of 3.
a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I.
value of 3 on the LLNA dose response plot.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period.
The animals did not show any clinical signs of toxicity after the in both experiments. Only in the first experiment after the third application the high dose (25%) induced redness of the ear skin of one animal of the group.

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 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item T002713 was found to be a skin sensitizer under the described conditions (S.I. > 3). The substance needs to be classified as skin sensitizer category 1B according to the CLP regulation (EC value > 2%).