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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
(2S,3S)-2-benzhydryl-N-[(5-tert-butyl-2-methoxy-phenyl)methyl]quinuclidin-3-amine
EC Number:
831-973-7
Cas Number:
147116-67-4
Molecular formula:
C32H40N2O
IUPAC Name:
(2S,3S)-2-benzhydryl-N-[(5-tert-butyl-2-methoxy-phenyl)methyl]quinuclidin-3-amine
Test material form:
solid: granular
Remarks:
Granular white powder
Details on test material:
Granular white powder
Specific details on test material used for the study:
Sponsor's identification CJ-11,972
Description white solid
Batch number 52855-16-4
Date received I I April 2001
Storage conditions room temperature in the dark




In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals and environmental conditions:
Male BALB/c (BALB/cAnNCrlBR) strain mice were supplied by Charles River UK Limited.
Margate. Kent. UK. At the start of the study the animals weighed 18 to 21 g, and were
approximately six to eight weeks old. After an acclimatisation period of at least five days. each
animal was selected at random and given a number unique within the study which was written on
the tail using a black indelible marker pen.
The animals were housed in groups of four in suspended polypropylene cages fitted with stainless
steel mesh lids and furnished with softwood woodtlakes. Free access to mains tap water and food
(Rat and Mouse Expanded Diet No. I. Special Diets Services Limited. Witham. Essex. UK) was
allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to
25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered
not to have affected the purpose or integrity of the study. The rate of air exchange was
approximately fifteen changes per hour and the lighting was controlled by a time switch to give
twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.1, 1 and 10% w/v
No. of animals per dose:
four
Details on study design:
The method used was that described by Kimber L Hilton J and Weisenberger C ( 1989) "The
Murine Local Lymph Node Assay for Identification of Contact Allergens : A Preliminary
Evaluation of in situ Measurements of Lymphocyte Proliferation". Contact Dermatitis 21. 215-
220 and Basketter D A and Scholes E W (1992) Comparison of the Local Lymph Node Assay
with the Guinea Pig Maximisation Test for the Detection of a Range of Contact Allergens, Food
and Chemical Toxicology 30. 65-69.
Sixteen mice were used. twelve test and four control. The bodyweight of each animal was
recorded at the start and end of the study.

In a preliminary investigation the test material was found to be soluble at a maximum
concentration of I 0% in acetone/olive oil 4: 1 (v/v). Concentrations of I 0%. l % and 0.1 % wlv in
acetone/olive oil 4: I (v/v) were therefore chosen for the main study.
Groups of four mice were treated with the test material at concentrations of 0. I, I or I 0% w/v in acetone/olive oil 4: 1 (v/v). The mice were treated by daily application of 25 µI of the
appropriate concentration of the test material to the dorsal surface of each ear for three
consecutive days (Days 0. 1. 2). The test material formulation was administered using an
automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

Five days following the first topical application of the test material (Day 5) all mice were injected
via the tail vein with 250 µI of phosphate buffered saline containing 3H-methyl thymidine
(HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, Amersham Pharmacia Biotech) giving a total
of 20 µCi to each mouse.
Positive control substance(s):
mercaptobenzothiazole (CAS No 149-30-4)

Results and discussion

Positive control results:
Proliferation 1% w/v 1.73; 5% w/v 1.71; 25% w/v 3.97

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
control
Parameter:
SI
Value:
0.7
Test group / Remarks:
0.1% w/v
Parameter:
SI
Value:
1.8
Test group / Remarks:
1 % w/v
Parameter:
SI
Value:
10.6
Test group / Remarks:
10% w/v
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
vehicle: 434.4 dpm/node
0.1% w/v: 300.5 dpm/node
1% w/v: 766.5 dpm/node
10% w/v: 4593.7 dpm/node

DETAILS ON STIMULATION INDEX CALCULATION
A test/control ratio of greater than 3 was recorded for the I 0% w/v concentration of the test
material. A test/control ratio of less than 3 was recorded for the two remaining concentrations of the test material (0.1 and I% w/v)

EC3 CALCULATION
Not calculated

CLINICAL OBSERVATIONS:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals throughout the study period.

BODY WEIGHTS
Bodyweight changes of the test animals between Day O and Day 5 were comparable to those observed in the corresponding control group animals over the same period.

SKIN IRRITATION
No evidence of skin irritation was noted at the treatment sites of test or control animals.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test material was considered to be a sensitiser under the conditions of the test.