10-undecenyl 2-cyano-3,3-diphenylpropenoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The study was performed between 16 November 2009 and 08 December 2009.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

Date of inspection 15th September 2009 Date of signature 26th November 2009

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-cyano-3-(4-methoxyphenyl)-3-phenyl-, undecenyl ester

IUPAC Name:

2-Propenoic acid, 2-cyano-3-(4-methoxyphenyl)-3-phenyl-, undecenyl ester

Constituent 2

Reference substance name:

Undecenyl methoxycrylene

IUPAC Name:

Undecenyl methoxycrylene

Constituent 3

Reference substance name:

700-824-2

IUPAC Name:

700-824-2

Constituent 4

Reference substance name:

undec-10-en-1-yl 2-cyano-3,3-diphenylprop-2-enoate

EC Number:

700-604-0

Cas Number:

947701-81-7

Molecular formula:

C27H31NO2

IUPAC Name:

undec-10-en-1-yl 2-cyano-3,3-diphenylprop-2-enoate

Constituent 5

Reference substance name:

2-Propenoic acid, 2-cyano-3-(4-methoxyphenyl)-3-phenyl, 10-undecen-1-yl ester

IUPAC Name:

2-Propenoic acid, 2-cyano-3-(4-methoxyphenyl)-3-phenyl, 10-undecen-1-yl ester

Details on test material:

Details on suurogate material:
Sponsor's identification : Undecenyl methoxycrylene
Description : amber coloured viscous liquid
Batch number : ABP401608
Date received : 10 August 2009
Storage conditions : room temperature in the dark

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 Limited, Bicester, Oxon, UK.

- Age at study initiation:
Eight to twelve weeks old.

- Weight at study initiation:
At the start of the study the animals were in the weight range of 15 to 23 g.

- Housing:
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.

- Diet (e.g. ad libitum):
Free access to food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.

- Water (e.g. ad libitum):
Free access to mains tap water was allowed throughout the study.

- Acclimation period:
At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19 to 25°C

- Humidity (%):
30 to 70%,

- Air changes (per hr):
The rate of air exchange was approximately fifteen changes per hour

- Photoperiod (hrs dark / hrs light):
Lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary Screening Test: 25 µl of the undiluted test material
Main Test: 100%, 50% , 25% v/v in acetone/olive oil 4:1

No. of animals per dose:

Preliminary Screening Test: 1
Main Test: 12
Control: 4

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the undiluted test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.

- Compound solubility:
Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.

MAIN STUDY
- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the undiluted test material or the test material at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test material 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 2.0 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)

Statistics:

None recorded.

Results and discussion

Positive control results:

Introduction.
A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Method (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.

Test Material: α Hexylcinnamaldehyde, tech., 85%
Project number: 0039/1120
Study dates: 05 November 2009 to 11 November 2009

Methods.
A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone.

Results.
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
15 3.12 Positive

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

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 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 shown in Table 5.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Please see Table 2 for results.

Any other information on results incl. tables

Clinical Observations and Mortality Data:

Individual clinical observations and mortality data for test and control animals are given in Table 3.

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

Bodyweight:

Individual bodyweights and bodyweight changes for test and control animals are given in Table 4.

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

Table1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
100%	S-1	18	19	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table2              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	13754.46	1719.31	na	na
25	20674.70	2584.34	1.50	Negative
50	17523.85	2190.48	1.27	Negative
100	9952.07	1244.01	0.72	Negative

 

Table3              Individual Clinical Observations and Mortality Data

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
25	2-1	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0
50	3-1	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0
100	4-1	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table4              Individual Bodyweights and Bodyweight Changes

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	18	19	1
	1-2	17	18	1
	1-3	17	18	1
	1-4	17	18	1
25	2-1	19	19	0
	2-2	18	19	1
	2-3	18	18	0
	2-4	19	18	-1
50	3-1	18	18	0
	3-2	19	20	1
	3-3	18	18	0
	3-4	20	20	0
100	4-1	18	19	1
	4-2	19	19	0
	4-3	20	20	0
	4-4	16	16	0

0=      No signs of systemic toxicity

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

Table 5 Stimulation index

Concentration (%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
25	1.50	Negative
50	1.27	Negative
100	0.72	Negative

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

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

Executive summary:

Introduction. 

A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

§        OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

§        Method B42 Skin Sensitisation (Local Lymph Node Assay) of CommissionRegulation (EC) No. 440/2008

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the undiluted test material or the test material as a solution in acetone/olive oil 4:1at 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	1.50	Negative
50	1.27	Negative
100	0.72	Negative

Conclusion. 

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