4-allyl-2-methoxyphenyl acetate

Endpoint summary

Description of key information

In vivo: skin sensitizer Cat. 1B (WoE - LLNA, OECD 429, GLP, Rel. 2)

QSAR: Skin sensitiser Cat. 1B (WoE - QSAR models, rel.2)

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:

2-9 November 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 429 with deviations: tested only up to 50% (without rationale); no preliminary study conducted; no ear thickness measurements reported

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

tested only up to 50% (without rationale); no preliminary study conducted; no ear thickness measurements reported

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME 04609.
- Females - nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Animals were healthy and free from infections.
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 18-24 g
- Housing: Animals were group housed (5 per cage) upon receipt then 4 per cage when assigned to groups.
- Diet: Certified Rodent Chow 7012C, ad libitum.
- Water: Tap water, ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: None

ENVIRONMENTAL CONDITIONS
- Temperature: 22.2-26.7 °C
- Humidity: 22-44 %
- Photoperiod: 12 h light/12 h dark

- IN-LIFE DATES: 2-9 November 2004

Vehicle:

other: Diethyl phthalate/ethanol (3:1)

Concentration:

2.5, 5, 10, 25 and 50% (w/v) in Diethyl phthalate/ethanol (3:1)

No. of animals per dose:

4

Details on study design:

DOSE PREPARATION
On each day of dosing, the test item was prepared 2.5, 5, 10, 25 and 50% (w/v) in volumetric flasks by dissolving the appropriate amount of test item in the vehicle. All preparations were vortexed to mix. The test item dosing solutions were clear colorless liquids.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The criterion for a positive response is that one or more concentrations of a test item elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

TREATMENT PREPARATION AND ADMINISTRATION:
Mice were treated on the dorsal surface of both ears (25 µL/ear), once per day on Days 1, 2, and 3. Approximately 24 ± 2 h between applications of test item was maintained. On Day 6, the mice were injected i.v. with 20 µCi of 3H-thymidine in 250 µL of sterile saline. Five hours later the mice were euthanized by CO2 asphyxiation. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. The lymph nodes from each group were pooled and a single cell suspension was prepared. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8 °C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a β-scintillation counter.
Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI).

Positive control substance(s):

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

Statistics:

The evaluation of the equality of means for body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, a Dunnett's test was used to determine the degree of significance from the control means.
No statistical analysis was done on the Disintegration per Minute (DPM).

Positive control results:

Hexylcinnamaldehyde at 35 % induced skin sensitisation (SI = 3.45).

Key result

Parameter:

SI

Value:

1.19

Test group / Remarks:

2.5%

Key result

Parameter:

SI

Value:

1.07

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1.28

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

2.16

Test group / Remarks:

50%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
At termination, the lymph nodes from the mice treated with the test item at 50% were enlarged but appeared normal. The lymph nodes from the mice in the vehicle and the test item treated animals at all other levels were normal in size and appearance.

DISINTEGRATION PER MINUTE (DPM)
Exposure to test item at 0, 2.5, 5, 10, 25 and 50% (w/v) resulted in DPM of 286, 340, 307, 278, 366 and 618, respectively

STIMULATION INDEX
Exposure to test item at 2.5, 5, 10, 25 and 50% (w/v) resulted in stimulation indices of 1.19, 1.07, 0.97, 1.28, and 2.16, respectively.

CLINICAL OBSERVATIONS:
- There was no mortality and all animals appeared normal throughout the study.
- The application sites on the mice from the groups treated with the test item at 50% appeared wet on Days 3-5. There were no other findings. No edema or erythema was noted at the application sites on any of the mice.

BODY WEIGHTS
There were no statistically significant differences in body weights observed between any of the treatment groups.

None

Interpretation of results:

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

Conclusions:

The registered substance did not induce skin sensitisation in an in vivo test (LLNA) at concentrations up to 50% with a stimulation index (SI) of 2.16. This SI is considered to be high and it could be anticipated to be higher than 3 with higher doses. Moreover, the registered substance was predicted to be a skin sensitizer based on QSAR models (ToxTree, Toolbox and VEGA) and its metabolite eugenol is a known sensitser. Therefore, considering all the evidence, the registered substance is classified as a skin sensitiser Cat. 1B according to the Regulation (EC) No 1272/2008 (EC3 > 2%).

Executive summary:

A study was performed to assess the skin sensitisation potential of test item in the CBA/J strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

 

Six groups of 4 CBA/J female mice were treated on the dorsal surface of both ears once per day for 3 days with 2.5, 5, 10, 25 or 50% (w/v) of test item with the vehicle (Diethyl phthalate/ethanol (3: 1)). On Day 6, the mice were injected, i.v. with 20 µCi of ³H-thymidine in sterile saline. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were precipitated with 5% trichloroacetic acid (TCA) and the pellets counted in a β-scintillation counter to determine incorporation of the ³H-thymidine.

 

There was no mortality and all animals appeared normal throughout the study. The application sites on the mice from the groups treated with the test item at 50% appeared wet on Days 3-5. There were no other findings. No edema or erythema was noted at the application sites on any of the mice. At termination, the lymph nodes from the mice treated with the test item at 50% were enlarged but appeared normal. The lymph nodes from the mice in the vehicle and the test item treated animals at all other levels were normal in size and appearance. There were no statistically significant differences in body weights observed between any of the treatment groups.

Exposure to test item at 2.5, 5, 10, 25 and 50% (w/v) resulted in stimulation indices of 1.19, 1.07, 0.97, 1.28, and 2.16, respectively.

 

The historical positive control, Hexylcinnamaldehyde, gave a SI of 3.45, when tested at 35 % w/v. The test system was therefore considered to be valid.

 

Under the test conditions, the registered substance did not induce skin sensitisation at concentrations up to 50% with a stimulation index (SI) of 2.16. This SI is considered to be high and it could be anticipated to be higher than 3 with higher doses. Moreover, the registered substance was predicted to be a skin sensitizer based on QSAR models (ToxTree, Toolbox and VEGA) and its metabolite eugenol is a known sensitser. Therefore, considering all the evidence, the registered substance is classified as a skin sensitiser Cat. 1B according to the Regulation (EC) No 1272/2008 (EC3 > 2%).

This study have been considered sufficient, together with in silico data to classify the registered substance without further testing using a weight of evidence approach.

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

QSAR approach

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

06 December 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Remarks:

No QPRF report was provided as model output. Those predictions have been considered sufficient, together with in vivo data on the substance (LLNA), to classify the registered substance without further testing using a weight of evidence approach.

Justification for type of information:

1. SOFTWARE
- VEGA v1.1.4
- Danish QSAR Database
- OECD QSAR Toolbox v4.2
- Toxtree v3.1.0

2. MODEL (incl. version number)
- Skin Sensitisation model (CAESAR) (version 2.1.6) of VEGA 1.1.4
- SciQSAR, LeadScope and CASE Ultra of Danish QSAR Database
- Structural alerts for protein binding of OECD QSAR Toolbox v4.2:
Protein binding by OASIS
Protein binding by OECD
Protein binding potency Cys (DPRA 13%)
Protein binding potency GSH
Protein binding potency Lys (DPRA 13%)
Keratinocyte gene expression
Protein binding alerts for skin sensitisation according to the GHS
Protein binding alerts for skin sensitisation by OASIS
Protein binding potency h-CLAT
- Decision trees of Toxtree v3.1.0:
Skin sensitisation reactivity domains
Protein binding alerts

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILES code / CAS Number : CC(=O)OC1=C(C=C(C=C1)CC=C)OC / 93-28-7

Qualifier:

no guideline followed

Principles of method if other than guideline:

The Skin Sensitisation potential of the registered substance was predicted using available structural alert systems and Quantitative Structure-Activity Relationship (QSAR) models.

GLP compliance:

no

Justification for non-LLNA method:

Those predictions have been considered sufficient, together with in vivo data on the substance (LLNA), to classify the registered substance without further testing using a weight of evidence approach.

Key result

Parameter:

other: Classification

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

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

Conclusions:

The registered substance was associated with protein binding alerts with Toxtree and Toolbox and classified as a skin sensitiser by the VEGA QSAR model.
Its metabolite Eugenol was also associated with skin sensitisation alerts and is a known skin sensitiser.
Therefore, considering all the evidences, the registered substance is classified as a skin sensitiser Cat. 1B according to the Regulation (EC) No 1272/2008 (EC3 > 2%).
Those predictions have been considered sufficient, together with in vivo data to classify the registered substance without further testing using a weight of evidence approach.

Executive summary:

The registered substance was associated with protein binding alerts with Toxtree and Toolbox and classified as a skin sensitiser by the VEGA QSAR model.

Its metabolite Eugenol was also associated with skin sensitisation alerts and is a known skin sensitiser.

Therefore, considering all the evidences, the registered substance is classified as a skin sensitiser Cat. 1B according to the Regulation (EC) No 1272/2008 (EC3 > 2%).

Those predictions have been considered sufficient, together with in vivo data to classify the registered substance without further testing using a weight of evidence approach.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A study was identifed on the substance (Calvert Laboratories, 2005). In this Local Lymph Node Assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP, groups of four female CBA/J mice were topically applied with test material at 2.5, 5, 10, 25 or 50 % (w/v) in 3:1 diethyl phthalate/ethanol on dorsal surface of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group was treated similarly with 3:1 diethyl phthalate/ethanol alone and a positive control group was treated with hexyl cinnamic aldehyde (HCA, 35 % w/v). On Day 6, the animals were injected intravenously with 20 µCi of 3H-thymidine in sterile saline. Five hours later, animals were euthanized and the draining auricular lymph nodes were removed and precipitated with 5 % trichloroacetic acid (TCA). 3H-thymidine incorporation was quantified using a β-scintillation counter and disintegrations per minute (DPM) and stimulation index (Sl) were calculated for each dose group.

There was no mortality and all animals appeared normal throughout the study. The application sites on the mice from the groups treated with the test item at 50% appeared wet on Days 3-5. There were no other findings. No edema or erythema was noted at the application sites on any of the mice. At termination, the lymph nodes from the mice treated with the test item at 50% were enlarged but appeared normal. The lymph nodes from the mice in the vehicle and the test item treated animals at all other levels were normal in size and appearance. There were no statistically significant differences in body weights observed between any of the treatment groups.

 

Exposure to test item at 2.5, 5, 10, 25 and 50% (w/v) resulted in stimulation indices of 1.19, 1.07, 0.97, 1.28, and 2.16, respectively.

 

The historical positive control, Hexylcinnamaldehyde, gave a SI of 3.45, when tested at 35 % w/v. The test system was therefore considered to be valid.

The maximum concentration tested in this study was limited to 50% (w/v) because the objective was for a risk assessment and not a hazard assessment. Under the test conditions, the registered substance did not induce skin sensitisation at concentrations up to 50% ((SI = 2.16). However, this SI is considered to be high and is anticipated to be above 3 at higher doses and therefore would lead to skin sensitisation.

This conclusion is supported by QSAR predictions (ToxTree, Toolbox and VEGA). Additionally, its metabolite - eugenol - is a known sensitser (see the additional discussion below).

Therefore, considering all the evidences, the registered substance is classified as a skin sensitiser Cat. 1B with an EC3 > 2%.

Additional discussion:

Based on experimental data (Castro 2004), eugenyl acetate is readily hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, eugenol. Slow rate of hydrolysis of esters in skin correlates with their decreased sensitization potential.

As detailed in the IDEA workshop on pre- and pro-haptens in Fragrance, in vivo skin sensitisation data suggests that eugenyl acetate does not hydrolyse rapidly enough in skin therefore the substance should not be considered equivalent to eugenol with regard to sensitisation induction. Indeed, with EC3 values = 5.4%, eugenol is considered to be a moderate skin sensitizer (skin sensitizer Category 1B) (disseminated dossier) whereas eugenyl acetate does not have the potential to induce skin sensitization up to 50% (highest concentration tested). The comparison of SI is included in the Table below:

Concentration (w/v)	SI - Eugenyl acetate	SI - Eugenol
2.5	1.19	1.2
5	1.07	2.7
10	0.97	6.0
25	1.28	14.3
50	2.16	19.4

References:

- Castro DJ, Sweet CJ, Kuester RK and Sipes G. Hydrolysis of Isoeugenyl-acetate and Eugenyl-acetate by Rat and Human Hepatic Microsomes. 2004, Abstract No. 1446, The Toxicologist- a supplement to Toxicological Sciences, 78 (S-1), 298. Cf. IUCLID section 7.1.1.

- IDEA Workshop - Risk assessment of pre- & pro-haptens (May 28-29, 2013). "Part 2 - Hydrolysis" and "The in vitro hydrolysis of isoeugenyl acetate and eugenyl acetate". Cf. IUCLID section 7.1.1.

- Eugenol disseminated dossier: https://echa.europa.eu/registration-dossier/-/registered-dossier/13694/1

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, the substance is classified as Skin Sens. 1B (H317: May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No data was available for respiratory sensitisation.