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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:
21 April 2004 to 05 May 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
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:
-
EC Number:
451-620-7
EC Name:
-
Cas Number:
352230-22-9
Molecular formula:
Constituent 1: C18H28O2Si3 Constituent 2: C30H38O3Si4
IUPAC Name:
Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Stability under test conditions: stable under test conditions
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was placed in a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogeniser. The test formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, the Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16-24 g
- Housing: Individually in Makrolon type-2 cages with standard softwood bedding
- Diet: pelleted standard mouse maintenance diet, ad libitum
- Water: community tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Indication of any skin lesions: none reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 30-70%
- Air changes (per hour): 10-15
- Photoperiod (hours dark / hours light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25 and 50% in acetone/olive oil, 4:1 (v/v) and 100% (undiluted)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: yes. A non-GLP solubility test was performed where a suitable vehicle was chosen.
- Irritation: yes. A non-GLP pre-test was performed in two mice. The test item was tested at four different concentrations 10, 25, 50 in acetone/olive oil and 100%. It was determined that 100% was the highest technically applicable concentration whilst avoiding systemic toxicity.
- Systemic toxicity: yes
- Ear thickness measurements: no
- Erythema scores: no

MAIN STUDY
Each test group was treated by topic application to the dorsal surface of each ear lobe with different test item concentrations of 25, 50 % in acetone/olive oil and 100% undiluted. The application volume was 25 µl. It was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A control group was treated with the vehicle only. A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied.
250µl of 81.5 µCi/ml 3H-Methyl-thymidine was administered to all animals by intravenous injection via a tail vein five days after the first topical application.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin Sensitisation: Local Lymph Node Assay
- Criteria used to consider a positive response: 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 stimulation index. Second, the data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: Dose formulations were prepared at least 4 hours prior to application. The test material was applied topically to the dorsal surface of each ear.
Statistics:
Not used

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
25% in acetone/olive oil
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
50% in acetone/olive oil
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
100%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: Background 3HTdR levels were measured in two 1ml-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdRmeasured on a β-scintillation counter.

DETAILS ON STIMULATION INDEX CALCULATION: Not specified

EC3 CALCULATION: Calculation of EC3 value was not done because no test concentration produced a stimulation index of 3 or higher.

CLINICAL OBSERVATIONS: No deaths occurred during the study. No clinical signs were observed in any of the animals of the control group or 25% treatment group. On the second application day, a slight ear swelling was observed at both dosing sites in all mice of 100% group, persisted for a total of four days. On the third application day, a slight ear erythema was observed at both dosing sites in all mice of 50% and 100% groups, persisting for a total of 3 days.

BODY WEIGHTS: The recorded body weights of the animals were 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 Local Lymph Node Assay, conducted according to an appropriate OECD test guideline and in compliance with GLP, reported the test substance to be not a skin sensitiser.