Is Isoeugenol a Prehapten? Characterization of a Thiol-Reactive Oxidative Byproduct of Isoeugenol and Potential Implications for Skin Sensitization

Article abstract of DOI:10.1021/acs.chemrestox.9b00501

Isoeugenol is widely used by the cosmetic and fragrance industries, but it also represents a known cause of skin sensitization adverse effects. Although devoid of a structural alert, isoeugenol has been classified as prehapten in virtue of the presence of a pre-Michael acceptor domain. Isoeugenol oxidation could theoretically lead to the generation of reactive toxic quinones, and photoinduced oxidative degradation of isoeugenol was reported to generate strongly thiol reactive byproducts. Nonetheless, the isoeugenol degradation product responsible for increased reactivity was found to be elusive. In the present study, an aged isoeugenol sample was subjected to reactivity-guided experiments to trap elusive thiol reactive species with a fluorescent nucleophile, viz. dansyl cysteamine (DCYA). The results herein presented demonstrate that photo-oxidation of isoeugenol led to the formation of a dimeric 7,4′-oxyneolignan with strong chemical reactivity, capable of nucleophilic substitution with thiols. The results were confirmed by isolation, structural characterization, and further NMR reactivity studies. Isoeugenol is already well-known as moderately reactive in thiol depletion assays, and was herein demonstrated to be capable of converting to more potent electrophilic species upon degradation, thus acting as a prehapten. The application of the reactivity-guided strategy described herein was shown to serve as an effective tool to investigate elusive skin sensitizers.

Full text of DOI:10.1021/acs.chemrestox.9b00501

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Article
Is isoeugenol a pre-hapten? Characterization of a thiol-reactive oxidative
byproduct of isoeugenol and potential implications for skin sensitization.
Jongmin Ahn, Cristina Avonto, Amar G Chittiboyina, and Ikhlas A Khan
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00501 • Publication Date (Web): 02 Mar 2020
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Is isoeugenol a pre-hapten? Characterization of a thiol-reactive
oxidative byproduct of isoeugenol and potential implications for
skin sensitization.
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Jongmin Ahn†, Cristina Avonto†*, Amar G. Chittiboyina† and Ikhlas A. Khan†,‡
† National Center for Natural Products Research, Research Institute of Pharmaceutical Science, School of Pharmacy, The
University of Mississippi, University, MS 38677, United States; ‡ Division of Pharmacognosy, Department of BioMolecular
Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, United States.
*
Correspondence: cavonto@olemiss.edu; Phone: +1 662 915 1090; Fax: +1 662 915 7989.
Keywords: Skin sensitization, isoeugenol, chemical reactivity, in chemico methods, stability studies
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Isoeugenol is widely used by the cosmetic and fragrance industries, but it also represents a known
cause of skin sensitization adverse effects. Although devoid of a structural alert, isoeugenol has been
classified as pre-hapten in virtue of the presence of a pre-Michael acceptor domain. Isoeugenol oxidation
could theoretically lead to the generation of reactive toxic quinones, and photo-induced oxidative
degradation of isoeugenol was reported to generate strongly thiol reactive byproducts. Nonetheless, the
isoeugenol degradation product responsible for increased reactivity was found to be elusive. In the present
study, an aged isoeugenol sample was subjected to reactivity-guided experiments to trap elusive thiol
reactive species with a fluorescent nucleophile, viz. dansyl cysteamine (DCYA). The results herein
presented demonstrate that photo-oxidation of isoeugenol led to the formation of a dimeric 7,4'-
oxyneolignan with strong chemical reactivity, capable of nucleophilic substitution with thiols. The results
were confirmed by isolation, structural characterization, and further NMR reactivity studies. Isoeugenol is
already well-known as moderately reactive in thiol depletion assays and was herein demonstrated to be
capable of converting to more potent electrophilic species upon degradation, thus acting as a pre-hapten.
The application of the reactivity-guided strategy described herein was shown to serve as an effective tool
to investigate elusive skin sensitizers.
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Introduction
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Contact dermatitis is a chronic pathology that cannot be cured, but only mitigated by avoidance of
physical contact with the allergen. The timely identification of potential skin sensitizers is, therefore, an
important aspect in the development of safer formulations, for cosmetics and other topical products.
Fragrance ingredients are commonly found in everyday preparations, such as personal care, foods,
household, and industrial products, including insecticides, cleaners, and detergents. Some fragrance
ingredients are known to cause Allergic Contact Dermatitis (ACD) by acting as chemical sensitizers and
resulting in type IV delayed allergic reactions.1
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Isoeugenol (IE), one of the major constituents of clove essential oil, is broadly used by the cosmetic and
fragrance industries primarily due to its desirable sweet, spicy, woody, and carnation olfactory notes, but it
also represents a known cause of adverse effects, viz. skin sensitization. The occurrence of allergic reactions
to IE is so widespread that it has been included among the eight constituents of the fragrance mix I (FM I)
used in routine patch tests.2–4 Along with 23 other pure compounds and two natural extracts, IE has been
included among the 26 cosmetic fragrance ingredients which must be declared on the ingredients list in
commercial products regulated by the EU Cosmetics Directive.5
Skin sensitizers are predominantly small, lipophilic electrophiles that can trigger delayed allergic
reactions following recurring skin contact.6 Chemical sensitizers typically initiate the immune-eliciting
pathways leading to ACD by covalent binding to reactive nucleophilic amino acids in skin proteins. This
formation of covalent adducts is at the core of the haptenation process, which is considered the Molecular
Initiating Event (MIE) according to the Adverse Outcome Pathway (AOP) described for skin sensitization.7
At a first glance, the structural feature of isoeugenol lacks of a mechanistic electrophilic domain capable of
triggering the MIE. Indeed, although sensitization to isoeugenol has been mainly attributed to biotic
activation by skin enzymes,8 evidence has also been reported about isoeugenol being susceptible to abiotic
activation through rapid oxidation.9 The chemical reactivity of isoeugenol, observed versus both thio- and
amino-peptides, has been explained by Patlewicz et al. based on the presence of a pre-Michael acceptor
domain, which could generate reactive quinone derivatives upon oxidation.10 Electrochemical oxidation of
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IE resulted in the generation of multiple O-demethylated and hydroxylated species capable of adduct
formation with proteins.11 During a stability-thioreactivity correlation study, IE was identified as
moderately unstable under exposure to photo-induced oxidative conditions, resulting in about 40%
degradation within 100 days of exposure to air and light at room temperature.12 The crude aged isoeugenol
(AIE) was shown to change over time, with the photo-oxidized material being more thiol reactive, but until
now candidates responsible for the observed reactivity remained elusive.12,13
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The first in chemico method adopted by the OECD was the Direct Peptide Reactivity Assay (DPRA),
which aims to characterize the MIE by measuring the covalent binding of chemicals to skin proteins.14 The
DPRA relies on the classification of chemicals based on the quantification of unreacted peptides by HPLC-
UV. In June 2019, a second chemical method, the Amino acid Derivative Reactivity Assay (ADRA), has
been added to the list of validated non-animal alternatives. Both methods rely on low-throughput, indirect
quantification of reactivity and have not been validated for testing multi-component articles.14
A fluorescent high throughput method using a dansyl thiol (HTS-DCYATM) was developed as a
convenient strategy to improve the sensitivity and throughput of in chemico methods.15 The HTS-DCYATM
relies on the same rationale as DPRA, but it enables the direct quantification of the hapten-thiol adducts by
fluorescence detection. The optimized workflow is particularly convenient for rapid, simultaneous
screening of a large number of test articles in a short time frame. Also, integration of the fluorescence assay
with analytical techniques, such as LC-MS, can facilitate the rapid identification of reactive compounds by
trapping them as stable fluorescent adducts. Indeed, the DPRA method has been modified to Spectro-DPRA
invigorated from HTS-DCYATM rationale and utility of such method was applied to cosmetic ingredients.16
In the present study, a DCYA-reactivity guided strategy was applied to identify elusive compounds
responsible for the increased chemical reactivity of photo-oxidized IE. The aged IE mixture was subjected
to chemical reactivity experiments and reactive compounds were trapped by reaction with the fluorescent
DCYA. Resulting adducts were isolated, characterized, and potential reaction mechanisms associated with
the observed reactivity were further explained by experiments with the NMR-DCYATM method.
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Material and Methods
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1. Materials
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Isoeugenol 1 (CAS. 97-54-1, mixture of cis and trans) with 98% purity and reagents for synthesis were
purchased from Sigma-Aldrich (St. Louis, MO, USA). The fluorescent compounds, DCYA and DCYA
disulfide, were synthesized as described previously.15 Maleimide polymer-supported (SiliaBond®,
Maleimide, ≥0.75 mmol/g) was purchased from SiliCycle (Quebec City, Quebec, Canada). Standardized
buffer solution pH 10, microcentrifuge tubes and polypropylene solvent-resistant 96 well microplates were
purchased from Fisher Scientific (Suwanee, GA, USA). Silica gel flash cartridges (SNAP Ultra 100 g and
SNAP HP-Sil 10 g) were purchased from Biotage (Charlotte, NC, USA). Flash chromatography
purifications were performed using an Isolera Four system (Biotage). Isoeugenol was irradiated for 100
days12,13 by bubbling air at a constant flow rate and simultaneously exposing the samples to cool light
irradiation conditions (6 h day/night cycles) using a compact fluorescent light bulb (860 lm, 6500 K, 60 W
equivalent, Philips® Andover, MA, USA). The samples were constantly stirred at room temperature for the
entire duration of the study, then stored at -20 ˚C. Chemical analysis of the resulting aged isoeugenol was
performed by GC-MS.12
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2. HTS-DCYATM method
The chemical reactivity of test articles was investigated using the in chemico HTS-DCYATM method.15
Briefly, a 2.5 mM solution of DCYA was mixed with an equal volume of 5.0 mM of test samples (821
μg/ml for AIE) in acetonitrile. The resulting solution was then transferred into two microcentrifuge tubes
(150 μL in each tube). Aqueous pH 10 buffer (30 μL) was added to the reaction (R) tubes; acetonitrile (30
μL) was added to the second tube (blank, Bl) instead of buffer. DCYA controls were prepared by mixing
the 2.5 mM solution of DCYA to an equal volume of acetonitrile. The obtained DCYA solution (150 μL in
each tube) was then incubated with aqueous pH 10 buffer (30 μL) for negative control (NC), or with
acetonitrile (30 μL) for positive control (PC). The samples were incubated for 23 min with vigorous shaking
at room temperature. Afterward, the reaction solutions were added to the vial containing 5 mg of maleimide
resin and incubated for 45 min with vigorous shaking. Sixty microliters of the supernatant were diluted to
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a final volume of 500 μL with ACN. Forty microliters (in triplicate) of the diluted solutions and 120 μL of
ACN were put into a 96 well black with clear bottom microplate. Each plate was read in triplicate using
fluorescence end-point readings (excitation 350 nm, emission wavelength 520 nm, cutoff 420 nm). The
degree of reactivity is shown as the reactivity index (RI), corresponding to the estimated formation of
covalent DCYA adducts with the test article. The RI was calculated based on the average of nine data (three
readings/well in triplicate) as follows:
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Bl ― R
PC ― Bl
RI = 100 × 1 ―
―
(
)
PC ― NC
PC
3. Isolation and characterization of DCYA adducts
Five mL of 215 mM DCYA solution in ACN was mixed with the same volume of 585 mM AIE solution.
Aqueous pH 10 buffer (0.5 mL) was added dropwise and the reaction was stirred for one hour at room
temperature. The reaction mixture was concentrated under vacuum, brine solution was added and the
solution was extracted with ethyl acetate. The organic phase was treated over anhydrous Na2SO4, filtered,
and the filtrate was evaporated to yield a crude residue which was purified by flash chromatography on
silica gel. The sample was loaded on a SNAP Ultra 100 g silica cartridge (Biotage) and eluted with hexane:
ethyl acetate (5%-100%, stepwise gradient). The resulting fractions were dried and analyzed by NMR and
HPLC-DAD-MS.
4. Isolation and characterization of 7,4'-oxyneolignan
AIE (200 mg) was separated using a SNAP HP-Sil 10 g silica cartridge (Biotage) and eluted with 25%
ethyl acetate in hexane. The 7,4'-oxyneolignan 4a (70.4 mg, 32.3 %) was isolated as a yellow oil and the
structure was elucidated by 1D- and 2D- NMR experiments and mass spectroscopy.
5. HPLC-DAD-MS analysis
High performance liquid chromatographic analyses were performed using an Agilent 1290 Infinity
HPLC system equipped with a quaternary pump, autosampler, column compartment, photodiode array
detector, and Agilent Open Lab ChemStation data acquisition software (Agilent Technologies, Santa Clara,
CA, USA). An SB-C18 RRHD, 2.1x100 mm, 1.8 µm column was used (Agilent Technologies). Isoeugenol
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samples were characterized using the following method: the solvent system was composed of solvent A
(water) and B (ACN) both containing 0.2% acetic acid; the flow rate was set at 0.2 mL/min. The gradient
used for characterization of isoeugenol samples was 20% to 45% B in 3 min then to 85% B in 15 min, to
90% B in 1 min hold for 3 min. UV was monitored at λ254nm. Positive and negative ESI spectra were acquired
from 100 to 1000 amu. Chemical analysis of DCYA experiments was performed by HPLC-MS using the
same conditions as above except for the following: 45% to 60% B in 3 min, then to 70% B in 17 minutes
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and to 90% B in 3 minutes, hold for 4 min. UV absorption profile was monitored at λ330nm
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6. NMR-DCYATM
The NMR-DCYATM assay was performed as described previously.17 Compound 4a was dissolved to 100
mM in CDCl3 (125 μL) and mixed with a solution 100 mM DCYA in CDCl3 (125 μL, containing 10 mM
of 2,5-dimethylfuran as the internal standard). A set of control 1H NMR spectra was recorded every 5 min
for approximately 70 min before addition of 1,5-diazabicyclo[4.3.0]non-5-ene (DBN). A 10 mM solution
of DBN in CDCl3 (25 μL) was then added, mixed and the reaction was analyzed by recording one spectrum
every 5 min for approximately 300 min. The spectra were processed using MNOVA 9.0 software. Depletion
of electrophilic signal (doEs) was calculated as follows:
∫퐶
100%
doEs =
∫퐼푆
With C = area of the signal of interest of the compound(s) and IS = area of the internal standard peak at
5.8 ppm.
7. m-Chloroperoxybenzoic acid (m-CPBA) oxidation
A solution of 2.56 mmol of 80% m-CPBA was added dropwise to a solution of isoeugenol (1.28 mmol)
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in CHCl3 at 0 C. The mixture was stirred at for 30 min and then brought to room temperature and stirred
for additional 1 h. Then the mixture was quenched with saturated NaHCO3 solution, diluted with distilled
water and extracted with CHCl3. The organic layer was separated, dried over anhydrous Na2SO4, evaporated
under reduced pressure and the resulting residue was purified by flash chromatography.
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8. tert-Butyl hydroperoxide (t-BHP) oxidation
A Solution of t-BHP (2.44 mmol, 70% in water or 5.0-6.0 M decane) was added dropwise to a stirred
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solution of isoeugenol (1.22 mmol) in CH2Cl2 at 0 C. The mixture initially was stirred at 0 C for 30 min
and then at room temperature. After 5h of stirring, the mixture was quenched with saturated NaHCO3
solution, diluted with water, extracted with DCM, dried over anhydrous Na2SO4 and concentrated under
reduced pressure to yield crude mixture.
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9. Epoxidation with N-Bromosuccinimide (NBS)
N-Bromosuccinimide (270 mg) in DME (4 mL) was added to a stirred solution of isoeugenol (250 mg)
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in 70% aqueous DME at 0 C portion wise. After 3 h of stirring, diluted with water (15 mL) and the mixture
was extracted twice with CH2Cl2. The combined organic layer was washed with saturated NaHCO3 solution,
dried over anhydrous Na2SO4, filtered and the filtrate was concentrated. The resulting bromohydrin residue
was subjected to the next reaction without further purification. The crude residue was dissolved in methanol
(10 mL) and added to a stirred suspension of anhydrous potassium carbonate (630 mg) in methanol (10
mL). After stirring for 1 h, the solution was neutralized by addition of 2N HCl, extracted twice with EtOAc.
The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure. The resulting crude product was purified by flash chromatography.
Results
Isoeugenol thiol reactivity increases upon photo-oxidation.
In order to understand the plausible chemical transformation of pre-haptens during the forced photo-
oxidative conditions tested, IE and AIE were probed for their thiol reactivity using the HTS-DCYATM
method. The AIE was obtained upon photo-induced oxidative degradation, to accelerate the degradation
process in the absence of solvent. Pure isoeugenol was classified as sensitizer with moderate thiol reactivity
toward DCYA (Table 1), despite the absence of direct reactivity alerts. After degradation, the resulting AIE
showed about three times increase in fluorescence response compared to IE, although it was not clear
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whether fluorescence changes were related to increased concentration of IE-derived activated species or to
the generation of different, more reactive byproducts.
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Table 1. HTS-DCYATM reactivity of isoeugenol samples after 23 min incubation.
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Samples
Reactivity Index (RI)
21.6 ± 2
Isoeugenol (1)
Aged isoeugenol mixture (AIE)
Compound 4a (cis/trans)
62.9 ± 4
116.6 ± 5
Fluorescence-guided isolation of reactive AIE components.
To identify the elusive reactive compounds generated upon IE degradation, additional trapping
experiments with DCYA were carried out. The AIE was treated with DCYA in pH 10 buffer. The thin-
layer chromatography profile of the reaction mixture showed one major fluorescent spot in addition to the
ones corresponding to unreacted DCYA and its autoxidation byproduct (DCYA)2 (Figure S1). The reaction
mixture was purified by flash chromatography and the structures of the major adduct 2 and a minor adduct
3 (Figure 1) were determined using 1D- and 2D-NMR and HPLC-MS experiments (Table S1, Figures S2-
1
S13). The regioselectivity of adduct 2 was confirmed by HMBC correlation (Figure S8) between the H
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(3.36 ppm) of 7″, belonging to the IE moiety, and the C signal of the CH2S of cysteamine (31.4 ppm).
The yield of isolated major adduct 2 was approximately 15%, and those of unreacted DCYA, isoeugenol
and (DCYA)2 were 17.8, 26.5 and 12.2%, respectively. Based on thorough structural characterization by
NMR, adduct 2 could have resulted from nucleophilic attack of DCYA on 7,4'-oxyneolignan 4a, a dimeric
oxidation product of isoeugenol.18,19 The thioacetal derivative 3 could have resulted from vanillin as a result
of thioacetalization with two molecules of DCYA.
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OH
OH
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Figure 1. The structure of isoeugenol (1), and isolated DCYA adducts 2 and 3.
Based on the structures of these adducts, it was initially postulated that degradation of 1 may occur with
the generation of reactive endoperoxide or epoxide species. Oxidative degradation byproducts have been
often associated with skin sensitization adverse effects due to their reactive, electrophilic nature.20 It has
been previously observed that some terpenoids, such as geranial and linalool, and aromatic compounds such
as cinnamyl alcohol, produce epoxide or peroxide via radical mechanisms. In several instances, such
autoxidation byproducts have been linked to evidence of skin sensitization in both in vivo and in vitro
models.21–25 It has also been reported that isoeugenol spontaneously autoxidized to give dimers and vanillin
through hypothetical formation and cleavage of a dioxetane intermediate(s).26–28 Formation of aldehyde
derivatives of structurally similar compounds, such as anethole and eugenol, as a result of photochemical
oxidation, has also been reported.29
Attempts to prepare isoeugenol epoxide.
Based on known oxidation mechanisms for similar volatile organic components (VOC), and the isolated
DCYA adducts, a plausible degradation pathway was proposed as shown in Figure 2. To validate this
hypothesis, attempts to prepare isoeugenol epoxide were carried out using reported methodologies. To our
surprise, isolation of IE epoxide derivatives was found to be quite elusive. Classic epoxidation conditions29–
31
using m-chloroperoxybenzoic acid (m-CPBA) and t-butyl hydroperoxide (tBHP), as well as two-step
process with N-bromosuccinimide (NBS) and alkali on isoeugenol and acetylated isoeugenol failed to
provide the expected epoxides. Only a major adduct of isoeugenol with m-CPBA was produced, while
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reaction with NBS led to the formation of methoxylated phenylpropanoid and benzofuranoid as sole
byproducts (Figure S14). Epoxidation using the bulky peroxide, tBHP, as well as attempts of epoxidation
of the acetylated IE derivative did not yield the anticipated isoeugenol epoxide.
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The elusive nature of the epoxide, and its tendency to ring-opening in the presence of oxidizing agents,
led to the supposition that such oxygenated intermediate may be too unstable and can easily undergo
nucleophilic attack in the presence of nucleophiles, such as mCPBA or isoeugenol. Such unexpected results
led to the premise that the epoxide may be too reactive to accumulate as a stable byproduct in AIE. It was
nonetheless clear that some reactive, non-epoxy degradation byproduct was present in the AIE sample.
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H
N
O2S
SH
H3CO
O2
OH
N
DCYA
1
O2
O
O
H3CO
O
O
H
O
- C2H4O
or
H3CO
OCH3
H3CO
H3CO
OH
OH
OH
OH
and/or
OH
+DCYA
x
+ DCYA (2 eq.)
OCH3
OH
O
H3CO
OH
OH
H
+DCYA
N
OCH3
OH
N
H3CO
O2S
S
H
H
N
N
N
S
S
S
S
O2
O2
4a
4b
threo (major)
erythro (minor)
2
3
N
Figure 2. Proposed degradation pathway of isoeugenol and formation of DCYA adducts
Isolation and characterization of IE photo-oxidative byproducts.
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As observed in the H, 13C and 2D-NMR spectra of crude AIE mixture, at least two additional
compounds were identified along with isoeugenol (Figures S15, S16) as major component. This was
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expected as we have previously shown that isoeugenol underwent only partial degradation when subjected
to forced degradation conditions.13 AIE was therefore subjected to flash chromatography, and the 7,4'-
oxyneolignan 4a was isolated along with IE. From the NMR analysis (Table S2, Figure S17-S18),
compound 4a was identified as diastereoisomeric mixture (trans:cis = approximately 5:1).18,19,32 The OH
group at C8 was confirmed by COSY and HMBC correlations between the doublet at 0.99 ppm and the
multiplet at 4.05 ppm (Figure S19, S21). In the 1H NMR spectrum of AIE, the integral ratio of δH 6.12-6.02
(H-8 of 1 and H-8' of 4a) and δH 4.49-4.41 (H-7 of 4a) was shown as 3:1 (Figure S15), indicating that there
were 2:1 mixture of isoeugenol (1) and its oxidized dimer 4a along with several minor compounds including
4b.
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Although 4a did not contain structural alerts, testing using the HTS-DCYATM method showed maximal
reactivity in the fluorescence assay (Table 1).
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Detailed NMR-DCYATM experiments (Tables S3-S4 and Figures 3, S22, S23) were then performed in
order to gain a mechanistic understanding of the reaction of 4a in the presence of the nucleophilic thiol.33
Incubation of 4a in the presence of DCYA alone did not cause any reactivity in the control reaction, thus
ruling out a possible pro-oxidant effect of 4a on DCYA dimerization. The addition of DBN as a catalyst
resulted in rapid depletion of 4a with the generation of adduct 2 and isoeugenol. The stoichiometric
equivalent of this reaction appears to be 1:1:1:1 (depletion of 4a: depletion of DCYA: generation of DCYA
adduct 2: generation of isoeugenol (1), Figure 3B), although further validation with kinetic NMR
experiments will be required. In any case, the data obtained by NMR experiments, therefore, confirmed
that 4a, a major component generated presumably from isoeugenol under oxidative conditions, is indeed
very reactive with thiols via nucleophilic substitution. It is worth noticing that substitution of 4a with DCYA
occurs through the re-generation of one mole equivalent of isoeugenol as a byproduct and warrants further
experimentation to establish the possible reaction mechanism.
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Figure 3. Observed NMR-DCYATM experimental data with 4a and DCYA. A) Nucleophilic
substitution of DCYA on 4a. B) Relative ratios of depletion of DCYA and 4a as well as formation of
adduct 2 after addition of DBN over a period of 200 min. C) Arrayed 1H NMR spectra indicating
depletion of signal corresponding to 4a (in purple box, H-7, δH 4.49-4.42 ppm) after addition of DBN. D)
Arrayed 1H NMR spectra indicating increase in intensity of signal corresponding to 2 (in orange box, H-2,
δH 3.41-3.35 ppm) after addition of DBN.
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Discussion
Autoxidation of VOCs is a well-studied phenomenon, which may occur with the generation of
potentially harmful byproducts. Attention to the chemical instability of fragrances and the change in their
reactivity is therefore an important aspect to establish the safety of formulations with respect to proper
storage and handling conditions.
The present investigation demonstrated that the photo-induced oxidation of isoeugenol predominantly
generates a 7,4'-oxyneolignan 4a with strong chemical reactivity. Isoeugenol is already well-known as
moderately reactive in thiol depletion assays12, and it can be activated to more potent electrophilic species
by oxidation, thus acting as a pre-hapten.20
Aliphatic terpenoids are especially prone to photo-oxidation via radical mechanisms. Oxidative by-
products such as conjugated aldehydes, peroxides, peroxyradicals, endoperoxides, and epoxides have been
linked to increased skin sensitization adverse effects of aged geraniol, linalool, and cinnamyl alcohol among
others.21,22,24,25 Interestingly, in the case of isoeugenol, typical epoxidation procedures failed to provide
stable products via chemical synthesis. Focused isolation of major degradation byproducts of IE resulted in
the identification of a strongly reactive 7,4'-oxyneolignan with no structural alert for skin sensitization.
Regardless of the lack of mechanistic domains, the compound was clearly reactive toward DCYA as model
nucleophile. Based on the data herein presented, it is therefore plausible that IE oxidative byproduct 4a
could trigger the initial key events leading to skin sensitization.
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It is worth notice that several authors reported the dimerization of IE to 8,4'-oxyneolignan(s) along or in
lieu of the 7,4'-oxyneolignan derivative herein described.18,27 Based on experimental evidence, it seems that
reaction conditions play a pivotal role in the dimerization process. The presence and protic nature of the
solvent, along with the choice of radical initiators, may constitute experimental factors critical to the fate
of IE photo-oxidative reactions.
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It is also important to mention in vivo data reported previously4 on skin sensitization potential of the 8-
O-4' regioisomer. Takeyoshi et al. reported that the 8,4'-oxyneolignan was only weakly reactive in guinea
pig maximization tests and caused no lymph node cell proliferation in LLNA experiments. The 8-O-4'
regioisomer was not isolated in aged isoeugenol samples, nor any thiol adduct of the same was identified.
Based solely on thiol trapping experiments on IE photo-oxidized in the absence of solvents, it seems that
the 7,4'-oxyneolignan may be of higher toxicological concern than its regiomeric analog.
Haptenation events are a fundamental KE in skin sensitization pathways; however, elicitation of
inflammatory pathways in keratinocytes and dendritic cells should follow in order to lead to clinical
outcomes. Further in vitro experiments will be required to determine whether such cellular cascades are
also activated upon exposure to 4a. More kinetic experiments will also be performed to further characterize
the mechanism of isoeugenol sensitization.
Trace amounts of 4a were also found in commercial lots of pure isoeugenol, which were stored in dark
glass bottles at 4 ˚C for an extended period of time. These observations confirmed findings reported
previously by Oka et al.19 The authors also observed that small traces of 4a are sufficient to antagonize the
response to isoeugenol in vitro by inhibiting the murine olfactory receptor mOR-EG. Prolonged storage in
the presence of an oxygen source could thus be sufficient to generate compounds of toxicological and
organoleptic concern. This is especially relevant in the cosmetic and fragrances industry, where high
volumes of pure chemicals are likely stored during the manufacturing process. Therefore, the formation of
reactive byproducts such as 4a is important for quality as well as for safety control of bulk materials under
good manufacturing practices.
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Acknowledgments
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This research was supported in part by “Science Based Authentication of Botanical Ingredients” funded
by the Food and Drug Administration, grant number 5U01FD004246-08.
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Abbreviation
ACD, Allergic Contact Dermatitis; ADRA, Amino acid Derivative Reactivity Assay; AIE, Aged
Isoeugenol; AOP, Adverse Outcome Pathway; DBN, 1,5-diazabicyclo[4.3.0]non-5-ene; DME, Dimethoxy-
ethane; DPRA, Direct Peptide Reactivity Assay; GC-MS, Gas Chromatography coupled with Mass
Spectrometry; HPLC-MS, High-Performance Liquid Chromatography coupled with Mass Spectrometry;
HTS-DCYATM, High Throughput Screening using Dansyl Cysteamine; IE, Isoeugenol; KE, Key Event;
MIE, Molecular Initiating Event; NMR, Nuclear Magnetic Resonance; OECD, Organisation for Economic
Co-operation and Development; RI, Reactivity Index; VOC, Volatile Organic Compounds.
Supporting Information
The NMR data of adducts, isolated IE byproducts and aged isoeugenol mixture, epoxidation attempts and
detailed data from NMR-DCYATM experiments were supplied in the supporting information.
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