Reaction mass of (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl palmitate and (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl stearate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-990-9

EC Name:

-

Cas Number:

376588-17-9

Molecular formula:

C38 H71 N O6

In vivo test system

Test animals

Species:

mouse

Strain:

other: 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: 7 - 9 weeks
- Weight at study initiation: 17.2 - 21.1 g
- Housing: In groups of four in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 3433, batch no. 84/02 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum.
- Water: Community tap water from Itingen, available ad libitum
- Acclimation period: 5 days

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

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10 %, 25 % and 50 % (w/w) in acetone:olive oil, 4:1 (v/v).

No. of animals per dose:

4

Details on study design:

RANGE FINDING TEST:
In a non-GLP conform pre-test in two mice, test item concentrations of 5 %, 10 %, 25 % and 50 % (w/w) were tested on one ear each. No irritation effects were observed at these concentrations after a single application. 50 % (w/w) was the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation in the chosen vehicle.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a glass beaker on a tared Mettler balance and the vehicle (acetone:olive oil, 4:1 (v/v)) was quantitatively added. The weight/weight dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment withthe magnetic stirrer. The preparations were made freshly before each dosing occasion.

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 10 %, 25 % and 50 % (w/w) in acetone:olive oil, 4:1 (v/v). 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). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied. Five days after the first topical application, all mice were administered with 250 µl of 85.3 µCi/ml 3H-METHYL THYMIDINE (3HTdR) (equal to 21.3 µCi 3HTdR) by intravenous injection via a tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zurich). 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 two times 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 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a p-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 (DPM).

- 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, 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.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 10%: 2.5 25% : 2.9 50%: 6.2 EC3 = 25.8 %

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per lymph node: Control Group: 607 10%: 1516 25% : 1738 50%: 3758

Any other information on results incl. tables

No test item-related clinical signs were observed in any animals of group 1 (control), group 2 (10%) or group 3 (25%). On the second application day, a slight ear swelling was observed at both dosing sites in all mice of group 4 (50 %), persisting for the remainder of

the in-life phase of the study. All treated animals survived the scheduled study period.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test item was found to be a sensitizer and an EC3 value of 25.8 % (w/w) was derived.

Executive summary:

Three groups each of four female mice were treated daily with the test item at concentrations of 10 %, 25 % and 50 % (w/w) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.

All treated animals survived the scheduled study period. In this study STIMULATION INDICES of 2.5, 2.9 and 6.2 were determined with the test item at concentrations of 10 %, 25 % and 50 % (w/w) in acetone:olive oil, 4:1 (v/v). The test item was found to be a sensitizer and an EC3 value of 25.8 % (w/w) was derived.