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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 31, 2009 to April 29, 2009
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

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

Constituent 1
Chemical structure
Reference substance name:
N-methyl-1-(trans-4-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclohexyl)methanesulfonamide
Cas Number:
1208319-26-9
Molecular formula:
C15H23N5O2S
IUPAC Name:
N-methyl-1-(trans-4-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclohexyl)methanesulfonamide
Specific details on test material used for the study:
Test material name in the study: PF-03394197-00
Batch-No.: JFB80003
Purity: 97.17%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V.
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: 8- 12 weeks
- Weight at study initiation: 17.7-23.1 g
- Housing: Group housing (where relevant)
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24°C
- Humidity (%): 35-65%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10, and 25% (w/v)
No. of animals per dose:
5 females
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 % (w/v) solution in dimethylsulfoxide after warming to 37°C and vortexing.
- Irritation:To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 10 and 25% each on three consecutive days. In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on Day 7 after treatment begin. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY
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 ul, was spread over the entire dorsal surface (0 - 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).

ANIMAL ASSIGNMENT AND TREATMENT
- 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: The test item was placed into a volumetric flask on a tared balance and dimethylsulfoxide was quantitatively added. The different concentrations were prepared by serial dilution.
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.
The ANOVA (Dunnett-test) was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. Statistical significance was at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
5% w/v
Key result
Parameter:
SI
Value:
0.33
Test group / Remarks:
10% w/v
Key result
Parameter:
SI
Value:
0.59
Test group / Remarks:
25% w/v
Cellular proliferation data / Observations:
No deaths occurred during the study period.
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.
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:
GHS criteria not met
Conclusions:
The test item was not a skin sensitiser under the described conditions.
Executive summary:

The LLNA study is performed according to OECD Guideline 429 under GLP. In this study Stimulation Indices of 0.80, 0.33, and 0.59 were determined with the test item at concentrations of 5, 10, and 25% in dimethylsulfoxide. The test item was not a skin sensitiser under the described conditions.