prop-2-en-1-yl (2E)-3-phenylprop-2-enoate

Endpoint summary

Description of key information

Skin sensitisation (OECD TG 429): Sensitising 1B

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2007 - 13 March 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This analogue information is used for read across to Allyl cinnamate

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca strain mice were supplied by Charles River UK Ltd., Manston Road, Margate, Kent, UK. 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. At the start of the study the animals were in the weight range of 18.1 to 23.7 g, and were eight to twelve weeks old.

The animals were group housed in cages suitable for animals of this strain and weight range. Free access to mains water and food (RM1, Special Diet Services Limited, Witham, Essex, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 22 ± 3°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 minimum 15 changes per hour and the lighting was controlled to give twelve hours continuous light 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:

other: 1:3 ethanol:diethylphthalate

Concentration:

Undiluted test item or the test item at concentrations of 0.5%, 1%, 2.5%, 5% or 10% in vehicle.

No. of animals per dose:

Groups of four mice were treated

Details on study design:

Preliminary Screening Test
Not performed. Dose levels for the main test were set by the sponsor in accordance with acute oral toxicity data provided.

Main Test
Test Item Administration
Groups of four mice were treated with the undiluted test item or the test item at concentrations of 0.5%, 1%, 2.5%, 5% or 10% v/v in 1:3 ethanol:diethylphthalate. 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. 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 a variable volume micropipette and spread over the dorsal surface of the ear. A further group of four 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, 5%, 10% or 25% w/v preparation of Hexylcinnamaldehyde in acetone in olive oil (4:1) was applied, and a vehicle control group was similarly treated using acetone in olive oil (4:1) alone.

3H-Methyl Thymidine Administration
Three days after the third application, all animals were injected via the tail vein with approximately 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:20µCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed at least once daily for signs of toxicity. Animals were monitored for any signs of irritancy on the ears on days 1, 2, 3 and 6 of the study. Irritancy was scored at a visual level with the scoring scheme of none detectable, mild, moderate or severe.

Bodyweights: The bodyweight of each animals was recorded prior to dosing on Day 1 and prior to injection of 3H methyl thymidine on Day 6.

Terminal Procedures
Termination: Approximately five hours following the administration of 3HTdR all mice were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

Preparation of Single Cell Suspension: A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200 mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3mL of 5% w/v Trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1mL of TCA.

Determination of 3HTdR Incorporation: The lymph node suspensions were transferred to scintillation vials and 10mL of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.

Data evaluation:
The results are expressed as a disintegrations per minute (dpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle group to give a test:control ratio known as the stimulation index (SI), for each concentration.
The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a skin sensitiser.

Positive control substance(s):

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

Statistics:

Not performed.

Positive control results:

The positive control item, Hexylcinnamaldehyde, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

Key result

Parameter:

EC3

Value:

3.1

Parameter:

SI

Value:

0.8

Remarks on result:

other: 0.5% test group

Parameter:

SI

Value:

1.3

Remarks on result:

other: 1.0% test group

Parameter:

SI

Value:

1.6

Remarks on result:

other: 2.5% test group

Parameter:

SI

Value:

7.5

Remarks on result:

other: 5% test group

Parameter:

SI

Value:

8.1

Remarks on result:

other: 10% test group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DETAILS ON STIMULATION INDEX CALCULATION
The following SI values were derived at 0.5, 1.0, 2.5, 5, and 10%: 0.8, 1.3, 1.6, 7.5 and 8.1, respectively.
EC3 CALCULATION
There was an indication that the test item elicits a SI ≥ 3 when tested up to 25%, Allyl phenoxyacetate was considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) is 3.1%.
CLINICAL OBSERVATIONS/IRRITANCY SCORING:
No mortality occurred. The application of the test substance did not result in an increase in visual levels of irritancy to the skin on or around the ear area apart from on day 3, where there was a slight reddening to the ear skin at 5 and 10% dose groups. This had resolved by day 6.

Interpretation of results:

other: Skin sensitiser.

Remarks:

According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The test item was considered to be a sensitiser under the conditions of the test and resulted in an EC3 of 3.1%.

Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 0.5, 1, 2.5, 5 and 10% the substance showed SI values of 0.8, 1.3, 1.6, 7.5 and 8.1, respectively. An EC3 has been derived resulted in an EC3 of 3.1%. Based on the results the substance needs to be classified as skin sensitiser category 1B and shall be labelled with H317: May cause an allergic skin reaction according to Regulation (EC) No. 1272/2008 and its amendments.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information

Justification for type of information:

The read across documentation is presented in the Skin sensitisation endoint summary. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Positive control results:

The positive control item, Hexylcinnamaldehyde, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

Key result

Parameter:

EC3

Remarks:

%

Value:

3.1

Parameter:

SI

Value:

0.8

Remarks on result:

other: 0.5% test group

Parameter:

SI

Value:

1.3

Remarks on result:

other: 1.0% test group

Parameter:

SI

Value:

1.6

Remarks on result:

other: 2.5% test group

Parameter:

SI

Value:

7.5

Remarks on result:

other: 5% test group

Parameter:

SI

Value:

8.1

Remarks on result:

other: 10% test group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DETAILS ON STIMULATION INDEX CALCULATION
The following SI values were derived at 0.5, 1.0, 2.5, 5, and 10%: 0.8, 1.3, 1.6, 7.5 and 8.1, respectively.
EC3 CALCULATION
There was an indication that the test item elicits a SI ≥ 3 when tested up to 25%, Allyl phenoxyacetate was considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) is 3.1%.
CLINICAL OBSERVATIONS/IRRITANCY SCORING:
No mortality occurred. The application of the test substance did not result in an increase in visual levels of irritancy to the skin on or around the ear area apart from on day 3, where there was a slight reddening to the ear skin at 5 and 10% dose groups. This had resolved by day 6.

Interpretation of results:

other: Skin sensitiser 1B

Remarks:

According to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

The test substance was considered to be a sensitiser, based on the results of the source substance.

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation potential of Allyl cinnamate is derived from Allyl phenoxyacetate The skin sensitisation executive summary of Allyl phenoxyacetate is presented first followed by the read-across rationale.

Allyl phenoxyacetate skin sensitisation LLNA:

The skin sensitisation potential of the source substance has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 0.5, 1, 2.5, 5 and 10% the substance showed SI values of 0.8, 1.3, 1.6, 7.5 and 8.1, respectively. An EC3 has been derived resulted in an EC3 of 3.1%. Based on the results the substance needs to be classified as skin sensitiser category 1B.

Allyl cinnamate (CAS no 56289-56-6) and its sensitising properties using read across from Allyl phenoxyacetate (CAS7493-74-5)

 

Introduction and hypothesis for the read across

Allyl cinnamate contains a phenyl-ring with an ethene-chain to which an ester with an allyl group is attached (see data matrix for chemical structure). For this substance no skin sensitisation data are available. Therefore additional information is used in accordance with Article 13 of REACH where it is said thatlacking information can be generated by i.e. applying alternative methods such as SARs, grouping and read-across.

Hypothesis:Allyl cinnamateis expected to have the same sensitising potential as its analogue Allyl phenoxyacetate based on similarity in structure and similar reactivity.

Available information:For Allyl phenoxyacetate a well conducted LLNA test (OECD TG 429; K1) is available, showing the presence of skin sensitising potential with an EC3 of 3.1%.

Target and Source chemical(s):The information on allyl cinnamate and the analogue information from allyl phenoxyacetate are presented in the data matrix.

Purity / Impurities:

The purity and impurities of the Allyl cinnamate do not indicate skin sensitisation potential other than indicated by the parent substance. The substance has purity close to 100% and all impurities are all below < 10%.

Analogue justification

According to REACH Annex XI an analogue approach and structural alert information can be used to replace testing when information from different sources provides sufficient evidence to conclude that this substance has or does not have a particular dangerous property. The result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.

Analogue selection: Analogues were searched for in the RIFM database (the communal database of the fragrance industry) and OECD QSAR toolbox. Only three substances from the OECD QSAR toolbox (Tanimoto similarity 60%) had the allyl =CH2 functional group present,vinyl cinnamate (CAS 3098-92-8), allyl phenoxyacetate (CAS 7493-74-5) and CAS 15814-45-6. Allyl phenoxyacetate (Tanimoto similarity 63%) is considered further as it contains an aromatic ring, an ester and a =CH2 group, which are also present in allyl cinnamate, and has relevant data.

Structural similarities and differences:Allyl cinnamate and Allyl phenoxyacetate are both allyl esters and contain a phenyl-ring. The structural difference between the two substancesis the double bond in the alkene chain attached to the aromatic ring in Allyl cinnamate, while Allyl phenoxyacetate has an oxygen (ether) bond there. This difference is further discussed below in the toxico-dynamic paragraph.

Toxico-kinetic, Dermal absorption: Allyl cinnamate and Allyl phenoxyacetate have similar molecular weights, are both liquids, have similar water solubilities and log Kow values indicating that these substances will be absorbed by the skin to a similar extent. Sensitisation is usually caused by the parent substance but also degradation products may cause skin sensitization.Metabolism: The ester is expected to be metabolized in the skin by carboxyl-esterases. The key metabolites are the phenyl-allyl (cinnamic) and phenoxyacid and allyl-alcohol.

Toxico-dynamics, Skin sensitisation reactivity:This allyl-alcohol is a known skin sensitizer and can be oxidized to its aldehyde. In view of both substances generating allyl-alcohol after metabolisation the skin the read across is justified. When the potency is considered theAllyl cinnamate is more stable compared to Allyl phenoxyacetate. This can be predicted because Allyl cinnamate has a higher LC50 for acute oral toxicity compared to Allyl phenoxyacetate: 1520 and 820 mg/kg bw, respectively and assuming that allyl-alcohol is the key toxic metabolite. Another measure is using the pKa which is a measure for the formation of the acid and the allyl alcohol. For Cinnamic acid and the Phenoxy acid the pKas are 4.12 (SPARC calculation) and 3.12 (internet source), respectively, indicating that the substance with the lower pKa will hydrolyse faster, thus Allyl phenoxyacetate. Therefore, the LLNA result of Allyl phenoxyacetate can be considered a conservative value for Allyl cinnamate.

Remaining uncertainties:There are no remaining uncertainties because Allyl phenoxyacetate is structurally a very close analogue with similar dermal absorption and reactivity.

Conclusions on skin sensitisation

For Allyl cinnamate no experimental skin sensitisation are available. Allyl phenoxyacetate is considered an analogue and its sensitisation information can be used for read across. When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation, which is presented in the current document. Allyl phenoxyacetate is a skin sensitizer with an EC3 of 3.1%, which can be used directly for read across to Allyl cinnamate. Therefore also Allyl cinnamate is a skin sensitizer 1B with the same potency.

Final conclusion: Allyl cinnamate needs to be classified as a skin sensitiser 1B.

 

Data matrix Information on Allyl cinnamate and Allyl phenoxyacetate supporting the read across for skin sensitisation.

CHEMICAL NAME	Allyl cinnamate	Allyl phenoxyacetate
	Target	Source
CAS	56289-56-6 (generic cas is1866-31-5)	7493-74-5
EINECS	813-349-6	231-335-2
REACH registration	REACH registered 2018	REACH registered
Molecular structure
Molecular weight	188.23	192.21
Physico-chemical properties
Appearance	Viscous liquid	liquid
Melting point (˚C) (EpiSuite)	29.72 (c) <-20 (m)	36.45 (c)
Vapour pressure at 25˚C(Pa) (EpiSuite)	0.002 (c) 0.46 (m)	1.08 (c)
Exp. Water solubility at 20˚C(mg/L) (EpiSuite)	59-92 (c) 97.2 (m)	148-380 (c)
Exp. Log Kow (Epistle)	3.2 (c) 3.4 (m)	2.46 (c)
Human health
Acute oral toxicity mg/kg bw	LD50: 1520 (OECD TG 401)	LD50: 820 (OECD TG 401)
Skin sensitisation animal test	Read across	EC3 3.1% (OECD TG 429)

 

Justification for classification or non-classification

Based on the results, the substance needs to be classified as skin sensitiser category 1B and shall be labelled with H317: May cause an allergic skin reaction according to EU CLP (EC No. 1272/2008 and its amendments).