5,6,7-TRIMETHYLOCTA-2,5-DIEN-4-ONE

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 Novembre 2003 to 12 december 2003

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)

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:

(2E,5Z)-5,6,7-trimethylocta-2,5-dien-4-one

Cas Number:

357650-26-1

Molecular formula:

C11H18O

IUPAC Name:

(2E,5Z)-5,6,7-trimethylocta-2,5-dien-4-one

Constituent 2

Reference substance name:

(2E,5E)-5,6,7-trimethylocta-2,5-dien-4-one

Cas Number:

847144-75-6

Molecular formula:

C11H18O

IUPAC Name:

(2E,5E)-5,6,7-trimethylocta-2,5-dien-4-one

Test material form:

liquid

Specific details on test material used for the study:

Batch No. 9000530064
Aspect: Slightly yellow liquid
Purity: 96.7%
Expiry date: 12-Sept-2004

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

CBA/Jlbm

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Services CH - 4414 FOllinsdorf / Switzerland
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16g - 24g
- Housing: Individually in Makrolon type-2 cages with standard softwood bedding.
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no. 54/03 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum.
- Water (e.g. ad libitum): Community tap water from ltingen, available ad libitum.
- Acclimation period: 7 days - Under test conditions after health examination.
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

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

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0.5, 2.5, 5, 10 % (test article)
5, 10, 25% (Positive control)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in two mice with concentrations of 10 %, 25 %, 50 % (w/v) and 100 % (undiluted) (pretest excluded from Statement of Compliance).
After a single application, no irritation effects were observed at the dosing sites applied at concentration of 10 %. At the observations of 4 hours after the topical application, a slight ear swelling was noted at the local sites dosed at 25 % and 50 % (w/v), and a moderate ear swelling at the site dosed at 100 % (undiluted).
10 % (w/v) was the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation in the chosen vehicle.


MAIN STUDY
- Irritation: No test item-related clinical signs were observed in any animals of the control group, Group 2 (0.5 %) or Group 3 (2.5 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (5 %) and Group 5 (10 %), persisting for a total of three days. In addition, on the second application day, a slight ear swelling was observed at both dosing sites in all mice of Group 5 (10 %), persisting for a total of three days.
- Systemic toxicity: None observed
- Ear thickness measurements: Not conducted

ANIMAL ASSIGNMENT AND TREATMENT
- 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.

TREATMENT PREPARATION AND ADMINISTRATION:
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 0.5 %, 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µI, was spread over the entire dorsal surface (0 - 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 µI of 76.6 µCi/ml 3HTdR (equal to 19.1 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 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).

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 group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.


Observations:
- Mortality/Viability: Twice daily
- Bodyweights: Prior to the first application and prior to necropsy
- Clinical Observations: Once daily
- Irritation: Once daily.
- Ear Thickness: Not conducted.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c

where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:

The test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a skin sensitizer and an EC3 value of 9.4 % (w/v) was derived.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

1.6

Results 2

Parameter:

SI

Value:

1.6

Test group / Remarks:

Groupe 2 / 0.5% (w/v) in acetone:olive oil 4:1 (v/v)

Results 3

Parameter:

SI

Value:

4.1

Test group / Remarks:

Groupe 3 / 2.5% (w/v) in acetone:olive oil 4:1 (v/v)

Results 4

Parameter:

SI

Value:

5.6

Test group / Remarks:

Groupe 4 / 5% (w/v) in acetone:olive oil 4:1 (v/v)

Results 5

Parameter:

SI

Value:

2.3

Test group / Remarks:

Groupe 5 / 10% (w/v) in acetone:olive oil 4:1 (v/v)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : See tables

DETAILS ON STIMULATION INDEX CALCULATION : see tables

EC3 CALCULATION :
EC3 = (a-c) [(3-d)/(b-d)] + c
EC3 = 9.4 % (w/v)

Where a: 5, b: 1.5, c:10 and d: 3.2

CLINICAL OBSERVATIONS:
No test item-related clinical signs were observed in any animals of the control group. Group 2 (5 %) or Group 3 (10 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (25 %), persisting for four days. On the third application day, a slight ear swelling was also observed at both dosing sites in all mice of Group 4 (25 %), persisting for the remainder of the in-life phase of the study

BODY WEIGHTS
The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Test item concentration %(w/v)		Measurement dpm	Calculation			Result
			Dpm - BG*	number of lymph nodes	dpm per lymph node**	S.I.
--	BG I	0	--	--	--	--
--	BG II	0	--	--	--	--
--	CG 1	1131	1131	8	141	--
0.5	TG 2	1858	1858	8	232	1.6
2.5	TG 3	4655	4655	8	582	4.1
5	TG 4	6364	6364	8	796	5.6
10	TG 5	2638	2638	8	330	2.3

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

CG= Control Group

TG = Test Group

S.I.= STIMULATION INDEX

*The mean value was taken from the figures BG I and BG II

**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

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

In this study STIMULATION INDICES of 1.6, 4.1, 5.6 and 2.3 were determined with the test item at concentrations of 0.5 %, 2.5 %, 5 % and 10 % (w/v), respectively, in acetone:olive oil, 4:1 (v/v).
A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).
The test item GR-85-2517 was found to be a skin sensitizer and an EC3 value of 1.6 % (w/v) was derived.

Executive summary:

In order to study a possible contact allergenic potential of GR-85-2517, four groups each of four female mice were treated daily with the test item at concentrations of 0.5 %, 2.5 %, 5 % and 10% (w/v) 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 CH-methyl thymidine). Approximately five hours after intravenous injection, the micewere sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in a ­ scintillationcounter.

No test item-related clinical signs were observed in any animals of the control group, Group 2 (0.5 %) or Group 3 (2.5 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (5 %) and Group 5 (10 %), persisting for a total of three days. In addition, on the second application day, a slight ear swelling was observed at both dosing sites in all mice of Group 5 (10 %), persisting for a total of three days.

All treated animals survived the scheduled study period.

The results obtained (STIMULATION INDEX (S.I.)) are reported in the following table. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

 

	Test item concentration %(w/v)	S.I.
Group 2	0.5 *	1.6 *
Group 3	2.5 *	4.1 *
Group 4	5	5.6
Group 5	10	2.3
EC3=1.6%(w/v)
* This value was used in calculation of EC3.

 

The results obtained and reported in the above table show a decreasing S.I. value at concentration of 10 %. As some test item related findings such as, slight ear erythema and swelling were observed at the local dosing sites, it might be interpreted by a hypothesis that dermal trauma may induce the migration of Langerhans cells from the skin to the draining lymph nodes. If these migrating Langerhans cells bear "environmental" antigens, then a low­ level proliferative response in the nodes may be induced. (Kimber I, Basketter D A. The murine local lymph node assay: a commentary on collaborative studies and new directions. Fd Chem Toxic 1992: 30: 165-1

In this study STIMULATION INDICES of 1.6, 4.1, 5.6 and 2.3 were determined with the test item at concentrations of 0.5 %, 2.5 %, 5 % and 10 % (w/v), respectively, in acetone:olive oil, 4:1 (v/v).

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

The test item GR-85-2517 was found to be a skin sensitizer and an EC3 value of 1.6%(w/v) was derived.