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Diss Factsheets

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:
2006-03-22 to 2006-04-05
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
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-hydroxyethyl)-1H,3H-quinazoline-2,4-dione
EC Number:
214-897-3
EC Name:
3-(2-hydroxyethyl)-1H,3H-quinazoline-2,4-dione
Cas Number:
1207-75-6
Molecular formula:
C10H10N2O3
IUPAC Name:
3-(2-hydroxyethyl)-1,2,3,4-tetrahydroquinazoline-2,4-dione
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00495665 RT001200G1A291
- Expiration date of the lot/batch: 2006-06-30
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (range of 20 ± 5 °C), light protected.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
- The test item was placed in a volumetric flask on a tared Mettler balance and the vehicle DMSO was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears.
Homogeneity of the test item in the vehicle was maintained until start of treatment using an appropriate homogenizer.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd. Laboratory Animal Service
- Age at study initiation: 8-12 weeks (at beginning of acclimatization)
- Weight at study initiation: 16-24 grams
- Housing: Individually in Makrolon type 2 cages with standard Lignocel softwood bedding.
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433 batch no. 76/05 mouse maintenance diet, ad libitum
- Water (e.g. ad libitum): Itingen community tap water, ad libitum
- Acclimation period:7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
-Other: music during at least 8 hours of the light period


Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
1, 3, and 10% (w/v)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
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: 2.5%, 5% and 10%, on both ears on three consecutive days.
- Compound solubility: In a non-GLP pre-test, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v), ethanol/water (7/3, v/v), ethanol/water (3/7, v/v), N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), propylene glycol, and polyethylene glycol 300. DMSO was found to be a suitable vehicle and was used in the main test. 10% was the highest technically achievable concentration in the chosen vehicle.
- Irritation: Neither clinical signs nor findings were oberved at the concentrations tested one day after each single application.
- Systemic toxicity: Neither clinical signs nor findings were oberved at the concentrations tested one day after each single application.
- Ear thickness measurements: no data
- Erythema scores: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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 test concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in the control mice, as indicated by the stimulation index (S.I.)
- Second, the data must be compatable with a conventional dose reponse, although allowance must be made (especially at higher 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 the test substance at concentrations of 1%, 3%, and 10% in DMSO. 25 uL was spread over the entire surface of each ear lobe once daily for three days. Another group of mice was treated with an equal volume of DMSO 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.

Results and discussion

Positive control results:
The SI values calculated for concentrations 5, 10 and 25% in acetone:olive oil, 4:1 (v/v) were 1.8, 2.9 and 6.2 respectively. Alpha-hexylcinnamaldehyde was therefore found to be a skin sensitizer in the LLNA tests and an EC3 value of 10.5% was calculated using linear interpolation.


In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
based on 4 animals of 1% w/v group in DMSO
Parameter:
SI
Value:
1.6
Test group / Remarks:
based on 4 animals of 3% w/v group in DMSO
Parameter:
SI
Value:
1.1
Test group / Remarks:
based on 4 animals of 10% w/v group in DMSO
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
dpm per lymhp node (pooled lymph nodes per dose group divided by the number of lymph nodes = 8):
1% w/v group: 646
3% w/v group: 1062
10% w/v group: 698

DETAILS ON STIMULATION INDEX CALCULATION: see results table above, no dose-response relationship was observed

EC3 CALCULATION: calculation of EC3 values was not performed because no test concentrations produced a S.I. of 3 or higher

VIABILITY/ MORTALITY
No deaths occurred during the study period.

CLINICAL OBSERVATIONS: No clinical signs 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 necropsy, was within the range commonly recorded for animals of the strain and age.

SIZE OF THE DRAINING LYMPH NODES
The size of the draining lymph nodes of Group 3 was 2-3 fold large compared to that of the control group. The size of the draining lymph nodes of Group 4 was 2 fold large compared to that of the control group.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study S.I. values of 1.0, 1.6 and 1.1 were determined with the test item at concentrations of 1%, 3% and 10%, respectively, in DMSO. T001200 was therefore found to be a non-sensitizer when tested up to the highest applicable concentration of 10% in DMSO.