Pent-1-ene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08.06.2010-23.06.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study under GLP with full documentation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

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:

Pent-1-ene

EC Number:

203-694-5

EC Name:

Pent-1-ene

Cas Number:

109-67-1

Molecular formula:

C5H10

IUPAC Name:

pent-1-ene

Details on test material:

- Lot/batch No.: 10023680
- Expiration date of the lot/batch: February 2011
- Purity: 99.3%
- Storage condition of test material: In the refrigerator (2-8 °C), light protected, under inert gas athmosphere

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 Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 19.4 - 23.3 g
- Housing: Single
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): The relative humidity in the animal room was between approximately 45 - 81 % (recommendation in OECD 423 guideline: 30 - 70%). This deviation to the study plan, however, does not affect the validity of the study.
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 25, 50, 100%

No. of animals per dose:

4 animals per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was 100% of the undiluted test item.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 50 and 100% each on three consecutive days.
- Lymph node proliferation 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.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: i) that exposure to at least one concentration of the test item resulted in an incorporation of3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index. ii) 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
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50, and 100% (w/w) in acetone:olive oil (4:1). The application volume, 25 µl, was spread over the entire dorsal surface (diameter ~8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application, all mice were administered with 250 µl of 82.0 µCi/ml 3HTdR (corresponds to 20.5 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. The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in 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 plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for each dose group.

Results and discussion

Positive control results:

0%: 4549 DPM
5%: 9263 DPM
10%: 15495 DPM
25%: 27949 DPM

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 25%: 0.83 50%: 0.82 100%: 0.58

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 0%: 4077 DPM 25%: 3400 DPM 50%: 3342 DPM 100%: 2377 DPM

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item 1-Pentene was not a skin sensitiser.

Executive summary:

In the study the test item Pentene-1 dissolved in acetone:olive oil (4:1) was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 25, 50, and 100%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 0.83, 0.82, and 0.58 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4 :1), respectively.

The test item Pentene-1 was not a skin sensitiser.