(Z)-hex-3-enyl benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

LLNA was performed before Oktober 2016

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 26 May 2016 and 22 June 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

(Z)-hex-3-enyl benzoate

EC Number:

246-669-4

EC Name:

(Z)-hex-3-enyl benzoate

Cas Number:

25152-85-6

Molecular formula:

C13H16O2

IUPAC Name:

(3Z)-hex-3-en-1-yl benzoate

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands.
On receipt the animals were randomly allocated to cages.
The animals were nulliparous and non pregnant.
After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card.
At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively.
The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Based on the anticipated sensitization potential, groups of five mice were treated with the undiluted test item or the test item at concentrations of 25% or 5% (v/v) in acetone/olive oil 4:1.

No. of animals per dose:

Three groups, each of five animals per dose. A further group of five animals was treated with acetone/olive oil 4:1 alone.

Details on study design:

Test Item Preparation and Analysis
For the purpose of the study, the test item was used undiluted and freshly prepared as a 25% or 5% v/v solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The vehicle determination record is included under 'Any other information on materials and methods incl. tables'. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Preliminary Screening Test
As no toxicological information was available 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 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. Local skin irritation was scored daily according to the scale shown below:
Scale for Erythema (observation and score)
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema: 4

Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), 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 were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test
Test Item Administration
Based on the anticipated sensitization potential, groups of five mice were treated with the undiluted test item or the test item at concentrations of 25% or 5% (v/v) in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. 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 three consecutive days (Days 1, 2, 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 group of five mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
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 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Data Evaluation
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal 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 item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer". The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:

EC3 = c + [[(3-d)/(b-d)] x (a-c)]
a = lowest concentration giving stimulation index >3
b = actual stimulation index caused by ‘a’
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by ‘c’

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used. Probability values (p) are presented as follows:
P<0.001: ***
P<0.01: **
P<0.05: *
P≥0.05: (not significant)

Results and discussion

Positive control results:

The strain of mouse used in these laboratories has been shown to produce satisfactory responses using known sensitizers and non-sensitizers during the in-house validation. The results of routine positive control studies are shown below:

Methods
Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at concentrations of 5%, 10% or 25% (v/v). A further group of five animals was treated with acetone/olive oil 4:1 alone and served as the vehicle control group.

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 shown under ‘Any other information on results incl. tables’. The concentration of α-Hexylcinnamaldehyde, tech., 85% expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 20%.

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

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

24

Remarks on result:

other: (%)

Results 2

Parameter:

SI

Remarks on result:

other: The following SI values were derived at 5, 25 and 100%:1.53; 3.11 and 3.83, respectively.

Results 3

Parameter:

other: NOAEC

Value:

5

Remarks on result:

other: (%)

Cellular proliferation data / Observations:

Preliminary Screening Test
Clinical observations, body weight and mortality data and local skin irritation is given under ‘Any other information on results incl. tables’. The ear thickness measurements and mean ear thickness changes are given under ‘Any other information on results incl. tables’. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information, and anticipated sensitization potential, the undiluted test item and the test item at concentrations of 25% and 5% (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
The radioactive disintegrations per minute per lymph node and the stimulation index are given under ‘Any other information on results incl. tables’. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group is shown under ‘Any other information on results incl. tables’.

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.

Body Weight
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Calculation of EC3 Value
EC3 = c + [[(3-d)/(b-d)] x (a-c)]
a = 25
b = 3.11
c = 5
d = 1.53
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 24%.

Any other information on results incl. tables

Positive Control - 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:

Treatment	Stimulation Index	Result
5% v/v in acetone/olive oil 4:1	1.42	Negative
10% v/v in acetone/olive oil 4:1	1.53	Negative
25% v/v in acetone/olive oil 4:1	3.71	Positive

Clinical Observations, Body Weight and Mortality Data – Preliminary Screening Test

Concentration	Animal Number	Body Weight (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	18.8	19.0	0	0	0	0	0	0	0	0	0

0 = No signs of systemic toxicity

Local Skin Irritation – Preliminary Screening Test

Concentration

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

100%

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
100%	S-1	0.21	0.20	0.23	0.20	0.22	0.20
overall mean (mm)		0.205		0.215		0.210
overall mean ear thickness change (%)		na		4.878		2.439

na = not applicable

Individual Disintegrations per Minute and Stimulation Index

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle	1-1	3495.11	2869.60 (±572.62)	na	na
	1-2	2152.92
	1-3	3283.14
	1-4	2405.32
	1-5	3011.49
5	2-1	5087.12	4386.32 (±1815.30)	1.53	Negative
	2-2	3068.22
	2-3	6130.62
	2-4	1915.59
	2-5	5730.06
25	3-1	9939.56	8915.78** (±2494.60)	3.11	Positive
	3-2	8898.63
	3-3	7109.97
	3-4	6132.34
	3-5	12498.41
100	4-1	7439.55	10994.00*** (±2590.81)	3.83	Positive
	4-2	11097.12
	4-3	14182.07
	4-4	12540.91
	4-5	9710.34

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

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

na = not applicable

** = Significantly different from control group p<0.01

*** = Significantly different from control group p<0.001

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
5	1.53	Negative
25	3.11	Positive
100	3.83	Positive

Applicant's summary and conclusion

Interpretation of results:

other: Skin sensitiser (Category 1B)

Remarks:

according to EU CLP (1272/2008 and its amendments)

Conclusions:

The test substance was considered to be a sensitizer under the conditions of the test and resulted in an EC3 value of 24%.

Executive summary:

The skin sensitization potential of the test substance has been tested according to OECD TG 429: “Local Lymph Node Assay” and in compliance with GLP principles. 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. Based on the anticipated sensitization potential, three groups, each of five animals, 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 25% or 5% (v/v). A further group of five animals was treated with acetone/olive oil 4:1 alone. At 5, 25 and 100% the substance showed SI values of 1.53; 3.11 and 3.83, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 24%. A NOAEC of 5% is derived.