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:

guideline study

Remarks:

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

Test material form:

not specified

Details on test material:

- 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 caging
- 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): 12/12 (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%: SI = 1
5%: SI = 2.04
10%: SI = 3.41
20%: SI = 6.14

EC3 = (a-c) [(3-d)/(b-d)] + c = 8.5 % (w/v)
a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot:
a = 5 (%)
b = 2.04 (SI)
c = 10 (%)
d = 3.41 (SI)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.83

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Value:

0.82

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

0.58

Test group / Remarks:

100%

Cellular proliferation data / Observations:

Mean DPM for 0, 25, 50 and 100% in AOO 4:1 were 4077, 3400, 3342 and 2377, respectively

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: No symptoms 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 treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Table 7.4.1/1: Calculation and Results of Individual Data

Test item concentration % (w/w)	Group	Measurement DPM	Calculation			Stimulation Index	Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)
---	BG I	17	---	---	---	---	---
---	BG II	*	---	---	---	---	---
---	1	4094	4077	8	509.6
25	2	3417	3400	8	425.0	0.83	Negative
50	3	3359	3342	8	417.8	0.82	Negative
100	4	2394	2377	8	297.1	0.58	Negative

Vehicle: acetone:olive oil (4:1)

DPM = Disintegrations per minute

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

* = due to technical reasons, value could not be used

The EC3 value could not be calculated, since all S.I.´s are below 3.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, 1-Pentene is not classified for skin sensitisation according to the annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

A study was performed to assess the skin sensitisation potential of Pentene-1 in the CBA/CaOlaHsd strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test with test material at concentrations of 50% in Acetone:Olive Oil (4:1) and 100% in which there were no signs of irritation or systemic effects toxicity up to 100%, this concentration was therefore selected as the highest dose investigated in the main test.

Three groups, each of four females, were treated with 50 μL (25 μL per ear) of test material at concentrations of 25, 50 and 100 % for 3 consecutive days. A further group of four animals was treated with acetone/olive oil 4:1 alone. The animals were allowed to rest without dosing on days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-Methyl Thymidine.

Stimulation index for 25, 50 and 100% w/w in acetone/olive oil 4:1 were 0.83, 0.82 and 0.58, respectively.

No EC3 value was calculated for test item as all stimulation index were lower than 3 at concentrations of 25 and 50 % v/v in acetone/olive oil 4:1.

There were no deaths. No signs of systemic toxicity or skin irritation were noted in the test or control animals during the test. Bodyweight of the animals was within the range commonly recorded for animals of this strain and age.

The positive control α-Hexylcinnamaldehyde gave a SI of 3.41 and 6.14 at 10 and 25%, respectively. The corresponding EC3 value for positive control is 8.5% (w/v). The test system was therefore considered to be valid.

Under the test conditions, test material is not classified as skin sensitizer according to the annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.