cis-hex-3-en-1-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 March 2012 to 27 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

cis-hex-3-en-1-ol

EC Number:

213-192-8

EC Name:

cis-hex-3-en-1-ol

Cas Number:

928-96-1

Molecular formula:

C6H12O

IUPAC Name:

hex-3-en-1-ol

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Cis-hex-3-en-ol, LFA
- Physical state: clear, colourless liquid
- Analytical purity: 98.59 %
- Lot/batch No.: 1Z50650
- Expiration date of the lot/batch: 06 June 2015
- Storage condition of test material: room temperature in the dark under nitrogen

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: the animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. The animals were provided with environmental enrichment which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

100, 50 and 25 %

No. of animals per dose:

4/dose

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/ irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for 3 consecutive days (Day 1, 2 3). The mouse was observed twice daily on Days 1, 2 and 3 and once on Days 4,5 and 6. Local irritation was scored daily. Any clinical of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on day 6.

The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6, any changes in the ear thickness were noted. Mean ear thickness changes increase of equal to or greater than 25 % was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for 3 consecutive days (Day 1, 2 and 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

A further 4 mice received vehicle alone in the same manner

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 20. Ci/ mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.31

Test group / Remarks:

25%

Remarks on result:

other: non-sensitising

Results 2

Parameter:

SI

Value:

1.52

Test group / Remarks:

50%

Remarks on result:

other: non-sensitising

Results 3

Parameter:

SI

Value:

1.74

Test group / Remarks:

100%

Remarks on result:

other: non-sensitising

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle: 8625.33; 25 %: 11305.86; 50 %: 13138.66 ;100 %: 15037.68

Any other information on results incl. tables

Preliminary screening test

No signs of systemic toxicity, visual skin irritation or irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted.

Based on this information the undiluted test item and the test item at concentrations of 50 % and 25 % v/v in acetone/ olive oil 4:1 were selected for the main test.

Main test

Estimation of the proliferative response of lymph node cells

Disintegrations per minute, disintegrations per minute/ node and Stimulation index

Concentration (% v/v) in acetone/ olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	8625.33	1078.17	na	na
25	11305.86	1413.23	1.31	Negative
50	13138.66	1642.33	1.52	Negative
100	15037.68	1879.71	1.74	Negative

dpm= Disintegrations per minute

a= Disintegrations per minute/ node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b= Stimulation Index of 3.0 or greater indicates a positive result

na= Not applicable

The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

Clinical observations and mortality data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group of animals over the same period.

Current positive control study for the local lymph node assay

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

Concentration (% v/v) in acetone/ olive oil 4:1	Stimulation Index	Result
25	5.76	Positive

α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The sensitising potential of the test item was assessed according to OECD guideline 429. The test item was considered to be a non-sensitiser.

Executive summary:

Introduction.

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the OECD Guideline No. 429 "Skin Sensitisation: Local Lymph Node Assay".

Methods:

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100% this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.

Three groups of four CBA/Ca strain mice were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/ olive oil 4:1 at concentrations of 50 % or 25 % v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone.

Results:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% v/v) in acetone/olive oil 4:1	Stimulation Index	Result
25	1.31	Negative
50	1.52	Negative
100	1.74	Negative

Conclusion:

The test item was considered to be a non-sensitiser under the conditions of the test.