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Administrative data

Description of key information

LLNA, not a skin sensitizer ( Read-across to Isoeugenyl acetate (reaction-mass of cis- and trans-isomers), OECD 429, GLP, K, Rel. 2)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 08 to 14, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study performed according to OECD test guideline No. 429 with some deviations: no ear thickness measurement. The study was not fully reliable but was considered sufficient for the purpose of risk assessment.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
no ear thickness measurement
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
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
- Age at study initiation: 10 weeks
- Weight at study initiation: 18-24 g
- Housing: Animals were housed in group of 4 per cage.
- Diet: Certified Rodent Chow 7012C, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23.3-23.9 °C
- Humidity: 16-36 %
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: December 08, 2004 To: December 14, 2004.
Vehicle:
other: Diethyl phthalate/ethanol (3:1)
Concentration:
1, 2.5, 5, 10 and 25 %
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: up to 25% (w/v) (the test item was not soluble at 50% w/w)
- Irritation: in general, the doses were selected so that the highest concentration maximizes exposure while avoiding excessive local irritation.
- Systemic toxicity: in general, the doses were selected so that the highest concentration maximizes exposure while avoiding systemic toxicity.
- Ear thickness measurements: not measured
- Erythema scores: not reported

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A substance is considered as a sensitiser if one or more concentrations of test material results in a 3-fold or greater increase in isotope incorporation relative to the vehicle control.
TREATMENT PREPARATION AND ADMINISTRATION:
- Dosing solutions were prepared on each day of dosing. All preparations were vortexed to mix.
- 25 µL of control or test material was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). Approximately 24 ± 2 h between applications of test material was maintained. On Day 6, animals were injected i.v. with 20 µCi of 3H-thymidine in 250 µL of sterile saline. 5 h later animals were euthanized by CO2 asphyxiation. 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.
- Disintegrations per minute (DPM) and stimulation index (Sl) were calculated for each dose group.
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.
Positive control results:
SI for the positive control substance hexyl cinnamic aldehyde was 5.80 which demonstrates the validity of this study.
Key result
Parameter:
SI
Value:
< 3
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
At termination, the lymph nodes from the mice in the vehicle and test material treated animals at all levels were normal in size and appearance.
DPM (as mean ± sem) for 0 (vehicle), 1, 2.5, 5, 10 and 25 % were 285, 304, 197, 184, 286 and 280, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for test material at 1, 2.5, 5, 10 and 25 % were 1.07, 0.69, 0.65, 1.00 and 0.98, respectively.

EC3 CALCULATION
As a SI of 3 or more was not recorded for all of the concentrations tested, test item was not considered to have the potential to cause skin sensitization. Thus no EC3 was calculated.

CLINICAL OBSERVATIONS:
No mortality or clinical signs were observed. No oedema or erythema was noted at the application sites on any of the animals.

BODY WEIGHTS
No statistically significant differences in mean body weight and body weight changes observed between any of the treatment groups.

None

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test material is not considered as skin sensitiser according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a Local Lymph Node Assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP, groups of female CBA/J mice (4 females/group) were topically applied with test material at the dose concentrations of 1, 2.5, 5, 10 and 25 % final concentration (25% w/v being the highest achievable concentration) in 3:1 diethyl phthalate/ethanol on dorsal surface of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group (4 females/group) was treated with 3:1 diethyl phthalate/ethanol alone and a positive control group (4 females/group) was treated with hexylcinnamic aldehyde (HCA) at the dose concentration of 5, 15 and 35 % in diethyl phthalate/ethanol (3:1) in same manner to confirm the sensitivity and reliability of the test method. On Day 6, animals were injected intravenously with 20 µCi of 3H-thymidine in sterile saline. 5 h 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.

No mortality, clinical signs or skin reactions were observed in any of the animals. No changes in mean body weights were observed. Mean DPM for 0 (vehicle), 1, 2.5, 5, 10 and 25 % were 285, 304, 197, 184, 286 and 280, respectively. SI calculated for test material was found to be 1.07, 0.69, 0.65, 1.00 and 0.98 for the dose concentrations of 1, 2.5, 5, 10 and 25 %, respectively, which indicates that test item did not show the potential to induce skin sensitization.

The SI for the positive control substance HCA was 5.80, which demonstrates the validity of this study.

Under the test conditions, test material is not considered as skin sensitiser according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity (cis- and trans-isomers).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is the trans isomer (E), as a mono-constituent substance. The source substance is a reaction mass, composed of two diastereoisomers (the source substance [trans] and its cis-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
The source and the target substances have a common major constituent (trans-isomer).
The study design (OECD 429, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material was clearly identified (purity = 98.7 % w/w) although the isomer ratio was not reported. It is however assumed to represent the source substance in terms of constituents and impurities. The result of the study [non-sensitizer] is adequate for classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the Local Lymph Node Assay conducted with the source substance is likely to accurately predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VII, 8.3.

4. DATA MATRIX
Cf. Iuclid Section 13. 
Reason / purpose:
read-across source
Reason / purpose:
data waiving: supporting information
Positive control results:
SI for the positive control substance hexyl cinnamic aldehyde was 5.80 which demonstrates the validity of this study.
Key result
Parameter:
SI
Value:
< 3
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
At termination, the lymph nodes from the mice in the vehicle and test material treated animals at all levels were normal in size and appearance.
DPM (as mean ± sem) for 0 (vehicle), 1, 2.5, 5, 10 and 25 % were 285, 304, 197, 184, 286 and 280, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for test material at 1, 2.5, 5, 10 and 25 % were 1.07, 0.69, 0.65, 1.00 and 0.98, respectively.

EC3 CALCULATION
As a SI of 3 or more was not recorded for all of the concentrations tested, test item was not considered to have the potential to cause skin sensitization. Thus no EC3 was calculated.

CLINICAL OBSERVATIONS:
No mortality or clinical signs were observed. No oedema or erythema was noted at the application sites on any of the animals.

BODY WEIGHTS
No statistically significant differences in mean body weight and body weight changes observed between any of the treatment groups.

None

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the available data on the source substance, the target substance is not considered to be skin sensitiser according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a Local Lymph Node Assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP, groups of female CBA/J mice (4 females/group) were topically applied with the source substance ( 2-Methoxy-4-prop-1-en-1-ylphenyl acetate) at the dose concentrations of 1, 2.5, 5, 10 and 25 % final concentration (25% w/v being the highest achievable concentration) in 3:1 diethyl phthalate/ethanol on dorsal surface of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group (4 females/group) was treated with 3:1 diethyl phthalate/ethanol alone and a positive control group (4 females/group) was treated with hexyl cinnamic aldehyde (HCA) at the dose concentration of 5, 15 and 35 % in diethyl phthalate/ethanol (3:1) in same manner to confirm the sensitivity and reliability of the test method. On Day 6, animals were injected intravenously with 20 µCi of 3H-thymidine in sterile saline. 5 h 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.

No mortality, clinical signs or skin reactions were observed in any of the animals. No changes in mean body weights were observed. Mean DPM for 0 (vehicle), 1, 2.5, 5, 10 and 25 % were 285, 304, 197, 184, 286 and 280, respectively. SI calculated for test material was found to be 1.07, 0.69, 0.65, 1.00 and 0.98 for the dose concentrations of 1, 2.5, 5, 10 and 25 %, respectively, which indicates that test item did not show the potential to induce skin sensitization.

The SI for the positive control substance HCA was 5.80, which demonstrates the validity of this study.

Based on the available data on the source substance, the target substance is not considered to be skin sensitiser according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No study was identified on the source substance (trans-Isoeugenyl acetate. However, three studies were available on the target substance ( Isoeugenyl acetate (reaction-mass of cis- and trans-isomers):

- a LLNA (Calvert, 2005, rel.2),

- a Guinea pig test (Itho, 1982, Rel.3), and

- a Human patch test (White, 1999, Rel.3).

The LLNA was the only reliable study and was therefore selected as the Key study.

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 the dose concentrations of 1, 2.5, 5, 10 and 25 % final concentration 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) (5, 15 and 35 %). 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.

No mortality, clinical signs or skin reactions were observed in any of the animals. No changes in mean body weights were observed. Mean DPM for 0 (vehicle), 1, 2.5, 5, 10 and 25 % were 285, 304, 197, 184, 286 and 280, respectively. SI calculated for test material were 1.07, 0.69, 0.65, 1.00 and 0.98 at 1, 2.5, 5, 10 and 25 %, respectively, which indicates that test item did not show the potential to induce skin sensitization.

The SI for the positive control substance HCA was 5.80, which demonstrates the validity of this study.

Under the test conditions, Isoeugenyl acetate is not considered as skin sensitiser.

Additional discussion:

Based on experimental data (Castro 2004), isoeugenyl acetate is rapidly hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, isoeugenol. 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 Isoeugenyl Acetate does not hydrolyse rapidly enough in skin therefore the substance should not be considered equivalent to Isoeugenol with regard to sensitisation induction. Indeed, with EC3 values between 0.5 and 3.8, Isoeugenol is considered to be a strong skin sensitizer (skin sensitizer Category 1A) (ECHA, 2016) whereas Isoeugenyl acetate does not have the potential to induce skin sensitization up to 25% (highest concentration tested, all SI < 1, no dose-relationship).

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.

IDEA Workshop - Risk assessment of pre- & pro-haptens (May 28-29, 2013). The in vitro hydrolysis of isoeugenyl acetate and eugenyl acetate.

ECHA (2016) Committee for risk assessment RAC Annex 2 Response to comments document (RCOM) to the opinion proposing harmonised classification and labelling at EU level of isoeugenol; [1]; (E)-2-methoxy-4-(prop-1-enyl)phenol; [2]; (Z)-2-methoxy-4-(prop-1-enyl)phenol; [3]; EC Number: 202-590-7; [1]; 227-678-2; [2]; 227-633-7; [3]; CAS Number: 97-54-1; [1]; 5932-68-3; [2]; 5912-86-7; [3]; CLH-O-0000001412-86-98/F, Adopted 10 March 2016.

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 on the supporting substance, no additional self-classification is proposed according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No data was available for respiratory sensitisation. However, this substance is not a skin sensitizer, therefore according to Figure R.7.3 -2 of the Chapter R.7 (V 4.1 - October 2015) the chemical is not considered as a respiratory sensitizer.