Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 17, 2007 to February 23, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
White to yellow tan colour
Specific details on test material used for the study:
Identity: CP-902684
Batch No.: E010005632 or 166-TNN-73
Aggregate state at room temperature: Solid
Colour: Tan
Purity: 98 %
Stability in solvent: Not indicated by the sponsor
Storage: At room temperature
Expiration Date: March 21, 2009

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system
Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age: 10 - 12 weeks (beginning of treatment)
Identification: Single caging. The animals were distributed into the test groups at random and identified by cage number.
Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
Environment: temperature 22 +3°C, relative humidity 30-70%, artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10 and 25 %
No. of animals per dose:
4 females (nulliparous and non-pregnant)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 25 % solution in dimethylsulfoxide.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 2.5, 5, 10, and 25 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
- Systemic toxicity: The test item in the main study was assayed at 5, 10, and 25%. The top dose is the highest technically achievable concentration; this concentration also avoided systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT : The animals were distributed into the test groups at random and identified by cage number. 4 animals were assigned per group. Animals were distributed to the respective dose groups starting with the vehicle control and continuing with increasing test item concentrations.
- Name of test method: LLNA
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- 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.

TREATMENT PREPARATION AND ADMINISTRATION: 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 dimethylsulfoxide. The application volume, 25 Rl, was spread over the entire dorsal surface (~ 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).
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.

Where appropriate, a statistical analysis will be conducted for assessment of the dose-response relationship, and the EC3 value will be 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:
Calculation
Measurement DPM DPM-BG No. of lympt nodes DPM per lymph node
5421 5384 8 672.9

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.29
Test group / Remarks:
5 % (w/v)
Key result
Parameter:
SI
Value:
3.03
Test group / Remarks:
10 % (w/v)
Key result
Parameter:
SI
Value:
6.32
Test group / Remarks:
25 % (w/v)
Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION

EC3 CALCULATION : the EC3 value is calculated according to the equation:
EC3 = (a-c) [(3-d)/(b-d)] + c
The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

CLINICAL OBSERVATIONS: The animals did not show any clinical signs of toxicity after the first and second application. After the third application the low dose (5%) induced slight redness of the ear skin of all 4 animals of the group. At higher concentrations a similar effect was not observed.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item CP-902684 was found to be not a skin sensitiser under the described conditions.
Executive summary:

In the study the test item CP-902684 dissolved in dimethylsulfoxide was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25%. The animals did not show any clinical signs of toxicity after the first and second application. After the third application the low dose (5 %) induced slight redness of the ear skin of all 4 animals of the group. At higher concentrations similar effects were not observed. In this study Stimulation Indices (S.I.) of 1.89, 1.86 and 2.21 were determined with the test item at concentrations of 5, 10, and 25% in dimethylsulfoxide, respectively. The test item CP-902684 was not a skin sensitiser in this assay.