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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:
2006-03-01 to 2006-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
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
Specific details on test material used for the study:
- Name of test material (as cited in study report): TIC2782 (T002907)
- Substance type: white solid
- Physical state: solid
- Analytical purity: 99.9%
- Purity test date: no data
- Lot/batch No.: 00467090 ( = charge 05C0836)
- Expiration date of the lot/batch: 2006-05-04
- Stability of test item: stable under storage conditions
- Storage condition of test material: at room temperature (range of 20 +/- 5 °C), light protected

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Remarks:
CaHsdRcc(SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 16 female (+ 3 females for pretest (non-GLP)) mice, CBA/CaHsdRcc (SPF) (nulliparous and non-pregnant); RCC Ltd, Laboratory Animal Servive, CH-4414 Füllinsdorf, Switzerland
- Age at beginning of acclimatization: 8-12 weeks
- Weight at treatment (day 1): 17.0 -21.3 grams
- Housing: Standard Laboratory Conditions; individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum):ad libitum, pelleted standard Kliba 3433, batch no. 76/05 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst).
- Water (e.g. ad libitum): ad libitum, community tap water from Itingen
- Acclimation period: at least 6 days, 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): 10-15
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light/12 hour dark cycle with at least 8 hours music during the light period

IN-LIFE DATES: From: 2006-03-08 To: 2006-03-13

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 10, 25 and 50% (w/v)
No. of animals per dose:
4 females per group; 3 test groups and 1 negative control group
Details on study design:
RANGE FINDING TESTS:
- In non-GLP solubility pretest, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v), ethanol/water (7/3, v/v) and N,N-dimethylformamide (DMF). N,N-dimethylformamide (DMF) was found to be a suitable vehicle and was selected and used in the main test. 50 % was the highest technically applicable concentration in the chosen vehicle.
- A non-GLP local toxicity pre-test was performed for determination of concentrations for the main test. Three single animals were each treated with one of three different concentrations: 10 %, 25 % and 50 %, in both ears on three consecutive days. No clinical signs were observed at these concentrations one day after each single application. One day after the third topical application, the residual test item was found at the dosing sites of 50 %.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with the test item at concentrations of 10 %, 25 % and 50 % in DMF. 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).
- Criteria used to consider a positive response: a test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled: first, 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 S.I.; second, 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.

ADMINISTRATION OF ³HTdR
- Five days after the first topical application, all mice were administered with 250 μl of PBS (phosphate buffered saline) containing 78.37 μCi/ml 3HTdR (equal to 19.6 μCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED ³HTdR
- Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2 (dry ice).
- The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing twice with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of ‘Irga-Safe Plus’ scintillation liquid and thoroughly mixed.
- The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated for the body weights.

Results and discussion

Positive control results:
Stimulation indices of 1.8, 2.9 and 6.2 were determined with the test item at concentrations of 5%, 10% and 25%, respectively, in acetone:olive oil, 4:1 (v/v).
Alpha-hexylcinnamaldehyde was therefore found to be a skin sensitizer in the LLNA test and an EC3 value of 10.5% was derived.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.7
Test group / Remarks:
10% w/v group (based on results of 4 animals)
Parameter:
SI
Value:
1.3
Test group / Remarks:
25% w/v group (based on results of 4 animals)
Parameter:
SI
Value:
0.9
Test group / Remarks:
50% w/v group (based on results of 4 animals)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
control group: 351 dpm per node (8 lymph nodes in total)
10% w/v group: 257 dpm per node (8 lymph nodes in total)
25% w/v group: 441 dpm per node (8 lymph nodes in total)
50% w/v group: 322 dpm per node (8 lymph nodes in total)

DETAILS ON STIMULATION INDEX CALCULATION
see Results

EC3 CALCULATION : no EC3 value could be calculated as the SI values for all groups were below 3

CLINICAL OBSERVATIONS:
No deaths occurred during the study period.
Neither clinical/local signs nor other findings were observed in any animals of the control group or Group 2 (10%). On the second application day, the residual test item was found at all the dosing sites in all mice of Groups 3-4, persisting for a total of two days.
No findings were observed on the size of the draining lymph nodes.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study S.I. of 0.7, 1.3 and 0.9 were determined with the test item at concentrations of 10 %, 25 % and 50 %, respectively, in DMF.
TIC2782 (T002907) was therefore found to be a non-sensitizer when tested up to the highest applicable concentration of 50 % in DMF.