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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
Study period:
From 23/09/2008 to 08/10/2008
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:
no
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: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-39453193-AAA (T003063)
- Physical state: solid (powder)
- Appearance: white to almost white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 507652
- Expiration date of the lot/batch: April 03, 2009 (retest date)
- Purity: 100,4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
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 treatment)
- Weight at study initiation: 19.7 - 24.5 g
- Housing: Single housing in a Makrolon Type I cage with wire mesh top; granulated soft wood bedding
- 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 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
The test item in the main study was assayed at 1, 10 and 25%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
No. of animals per dose:
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
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was a 25% dilution in dimethylformamide.
- Irritation: 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 animals did not show any signs of irritation or systemic toxicity. The test item in the main study was assayed at 1, 10 and 25%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Determination of incorporated 3HTdR
- Criteria used to consider a positive response: 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.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fullfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3 HTdR at least 3-fold or greater than that recorded in control mice, as indicated by stimulation index (SI). The estimated concentration of the test item required to produce a SI of 3 is referred to as the EC3 value.
- 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 dimethylformamide was quantitatively added. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.
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 1, 10, and 25% (w/v) in dimethylformamide. The application volume, 25 µL, was spread over the entire dorsal surface (diameter~ 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 µL of 78.6 µCi/mL 3HTdR (corresponds to 19.7 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing twice with phosphate buffered saline (approximately 10 mL), the lymph node cells were resuspended in 5% trichloroacetic acid (approximately 3 mL) and incubated (at ca. 4°C) for at least 18 hours for precipitation of macromolecules. Precipitates were resuspended oin 5% trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorportation was measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5% trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated (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:
The EC3 value was estimated to be < 5%. A precise calculation of the EC3 value was not possible since the S.I. was above the threshold of 3 even at the lowest tested concentration. The obtained values are within the expected range and the positive control experiment is considered fully valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.04
Test group / Remarks:
4 animals of 1% w/v group
Parameter:
SI
Value:
3.02
Test group / Remarks:
4 animals of 10% w/v group
Parameter:
SI
Value:
4.03
Test group / Remarks:
4 animals of 25% w/v group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
DPM per lymh node (8 lymph nodes in total):
1% w/v group: 547.3
10% w/v group: 808.8
25% w/v group: 1077.3

DETAILS ON STIMULATION INDEX CALCULATION
In this study Stimulation Indices (S.I.) of 2.04, 3.02, and 4.03 were determined with the test item at concentrations of 1, 10, and 25 % in dimethylformamide, respectively.

EC3 CALCULATION
EC3 = (a-c) [(3-d)/(b-d)]+c = 9.8% (w/v)
test group 2: 1% = a, S.I. of 2.04 = b
test group 3: 10% = c, S.I. of 3.02 = d
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. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

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 T003063 was found to be a skin sensitiser under the described conditions. The EC3 value calculated was 9.8% (w/v). As the EC3 value is > 2%, the substance is classified as skin sensitiser category 1B.