2-Butenoic acid, 1-ethyl-2-methylpropyl ester, (2E)-

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 15 November 2012 and 12 December 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were used. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five 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 to23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve 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.

Vehicle:

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

Concentration:

Preliminary Screening Test
50% or 25% v/v in acetone/olive oil 4:1
Main Test
50%, 25% or 10% v/v in acetone/olive oil 4:1

No. of animals per dose:

5

Details on study design:

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using two mice, one mouse per test item concentration. The mice were treated by daily application of 25 µl of the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were 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 included as attached Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The bodyweight of each mouse 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 were calculated between time periods Days 1 to 3 and Days 1 to 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 sensitisation.
Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% 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 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.
The positive control animals were similarly treated to the test animals except that 25 µl of the positive control item, α-Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

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:80µCi/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.
Bodyweights: The bodyweight 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 labelled with the project 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 ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by ß- scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty 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 twenty 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).

Positive control substance(s):

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

Statistics:

Statistical Analysis
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group meandisintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way
analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Positive control results:

Alpha-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of 10.19 which is greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.

Parameter:

SI

Remarks on result:

other: The stimulation index are 0.84, 1.13, 1.87 for test item at 10%, 25% and 50% v/v in vehicle, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The individual DPM's are 3657.61, 3074.95, 4122.52, 6843.39 and 37284.28 for vehicle, test item at 10%, 25%, 50% and positive control, respectively.

In the preliminary screening test, 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, the dose levels selected for the main test were 10%, 25% and 50% in acetone/olive oil 4:1.

In the main study,

Individual Disintegrations per Minute and Stimulation Indices are given in Table 1.

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 2.

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 3.

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.

Table 1              Individual Disintegrations per Minute and Stimulation Indices

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	3878.61	3657.61 (±881.17)	na	na
	1-2	2471.90
	1-3	4125.89
	1-4	4714.71
	1-5	3096.93
10	2-1	2825.94	3074.95 (±1309.14)	0.84	negative
	2-2	1843.58
	2-3	3926.89
	2-4	1913.75
	2-5	4864.61
25	3-1	3738.38	4122.52 (±1082.76)	1.13	negative
	3-2	2800.06
	3-3	3888.55
	3-4	4438.84
	3-5	5746.79
50	4-1	10690.91	6843.39 (±3925.81)	1.87	negative
	4-2	927.56
	4-3	9914.13
	4-4	7320.81
	4-5	5363.53
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	29939.46	37284.28*** (±9277.46)	10.19	positive
	5-2	28507.27
	5-3	45125.58
	5-4	49090.49
	5-5	33758.62

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<0.001

Table 2              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
	1-5	0	0	0	0	0	0	0	0	0
10	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
	2-5	0	0	0	0	0	0	0	0	0
25	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
	3-5	0	0	0	0	0	0	0	0	0
50	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
	4-5	0	0	0	0	0	0	0	0	0
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	0	0	0	0	0	0	0	0	0
	5-2	0	0	0	0	0	0	0	0	0
	5-3	0	0	0	0	0	0	0	0	0
	5-4	0	0	0	0	0	0	0	0	0
	5-5	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table 3              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	19	18	-1
	1-2	18	19	1
	1-3	19	20	1
	1-4	20	21	1
	1-5	19	19	0
10	2-1	16	18	2
	2-2	20	21	1
	2-3	19	19	0
	2-4	18	19	1
	2-5	20	19	-1
25	3-1	19	19	0
	3-2	20	21	1
	3-3	19	21	2
	3-4	19	20	1
	3-5	18	19	1
50	4-1	19	19	0
	4-2	18	18	0
	4-3	19	18	-1
	4-4	20	20	0
	4-5	16	16	0
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	18	18	0
	5-2	20	21	1
	5-3	21	19	-2
	5-4	19	20	1
	5-5	18	19	1

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

Executive summary:

The skin sensitisation potential of the test susbtance, TM 12 -209, was assessed according to OECD test guideline 429. No skin reaction was observed at test concentrations of 50%, 25% and 10% with an EC3 value more than 50% v/v, therefore the test substance is not a sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A GLP-compliant study, performed according to OECD Guideline 429 (Local Lymph Node Assay in mice), was available for assessment (Harlan, 2013). Four groups of five female mice were treated by topical application to the dorsal surface of each ear lobe with 0% (vehicle control), 10%, 25% and 50% solutions of Cosmofruit as supplied in acetone/olive oil (4:1 v/v) for three consecutive days. The test substance was not tested at 100% due to the irritation/corrosive concern based on the acrylate structure. A positive control group of five female mice was treated with hexyl cinnamic aldehyde (25% v/v in the same vehicle). Five days after the first topical application, the mice were injected intravenously (tail vein) with³H-thymidine. Five hours thereafter, the draining auricular lymph nodes were excised, pooled per animal, processed in physiologically-buffered saline, and analysed for lymphocyte proliferation by measuring the incorporation of³H-thymidine in a scintillation counter. The stimulation index (SI) was 0.84, 1.13, and 1.87 for the 10%, 25% and 50% solution of the test substance, respectively. The SI for the positive control was 10.19 and in agreement with a previous reliability check. Based on the results of this study, Cosmofruit is not considered to give a positive result in the LLNA test.

An EC3 value could not be derived but is considered to be >50%.

Migrated from Short description of key information:
In a LLNA, the skin sensitisation potential of the test susbtance, TM 12-209, was assessed according to OECD test guideline 429. No skin reaction was observed at test concentrations of 50%, 25% and 10% with an EC3 value more than 50% v/v, therefore the test substance is not a sensitizer.

Justification for selection of skin sensitisation endpoint:
The study was conducted on the target substance in vivo, in an appropriate test species and according to internationally recognised guidalines.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results in the LLNA study with EC3 >50%, Cosmofruit is not considered to give positive response to warrant classified for skin sensitisation, according to EU Directive 67/548/EEC. According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, no classification is needed for Cosmofruit on its skin sensitization potential.