Food and Chemical Toxicology 84 (2015) 8e17
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Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
Review
Safety and risk assessment of ceramide 3 in cosmetic products
Seul Min Choi, Byung-Mu Lee*
Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Seobu-ro 2066, Changan-ku, Suwon, Gyeonggi-do, 440-746, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 May 2015
Received in revised form
16 July 2015
Accepted 18 July 2015
Available online 21 July 2015
Ceramide 3 is used mainly as a moisturizer in various cosmetic products. Although several safety studies
on formulations containing pseudo-ceramide or ceramide have been conducted at the preclinical and
clinical levels for regulatory approval, no studies have evaluated the systemic toxicity of ceramide 3. To
address this issue, we conducted a risk assessment and comprehensive toxicological review of ceramide
and pseudo-ceramide. We assumed that ceramide 3 is present in various personal and cosmetic products
at concentrations of 0.5e10%. Based on previously reported exposure data, the margin of safety (MOS)
was calculated for product type, use pattern, and ceramide 3 concentration. Lipsticks with up to 10%
ceramide 3 (MOS ¼ 4111) are considered safe, while shampoos containing 0.5% ceramide 3 (MOS ¼ 148)
are known to be safe. Reported MOS values for body lotion applied to the hands (1% ceramide 3) and back
(5% ceramide 3) were 103 and 168, respectively. We anticipate that face cream would be safe up to a
ceramide 3 concentration of 3% (MOS ¼ 149). Collectively, the MOS approach indicated no safety concerns for cosmetic products containing less than 1% ceramide 3.
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
Ceramide 3
Cosmetics
Margin of safety (MOS)
Risk assessment
Contents
1.
2.
3.
4.
5.
6.
7.
8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Physical and chemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Production and usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Human exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.1.
Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.2.
Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.3.
Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.4.
Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Toxicological effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6.1.
General toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6.2.
Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6.3.
Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
6.4.
Immunotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
6.5.
Developmental/reproductive toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
6.6.
Local toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
6.7.
Ocular toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
6.8.
Other toxicities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Regulation status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Risk assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
8.1.
Hazard identification and doseeresponse assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
8.2.
Exposure assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
8.3.
Risk characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
* Corresponding author.
E-mail address: bmlee@skku.edu (B.-M. Lee).
http://dx.doi.org/10.1016/j.fct.2015.07.012
0278-6915/© 2015 Elsevier Ltd. All rights reserved.
S.M. Choi, B.-M. Lee / Food and Chemical Toxicology 84 (2015) 8e17
9.
Summary and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transparency document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
The stratum corneum is the outermost layer of the skin and
prevents water loss through the skin (Lampe et al., 1983). The
stratum corneum is composed of three lipid components: ceramides, cholesterol, and fatty acids (Bissett, 2009). Ceramides are a
family comprising distinct lipid molecular species characterized by
the introduction of hydroxyl groups on the acyl chain (Hannun and
Obeid, 2011; Novgorodov and Gudz, 2011). Ceramides play a key
role as components of the stratum corneum and also as second
messengers in various cellular events, including proliferation, differentiation, cell cycle arrest, apoptosis, and senescence (el Bawab
et al., 2002; Nikolova-Karakashian and Rozenova, 2010; Bikman
and Summers, 2011). The role of ceramides as a major regulator of
apoptotic cell death in a wide range of cell types, including lens
epithelial cells, cerebellar granule cells, and osteoblasts, has been
~ ano et al., 2008; Hill and Tumber,
investigated (Samadi, 2007; Min
2010). Other studies on ceramides have focused on their barrier
function in the skin, because decreased levels of ceramides within
the intercellular lipid lamellae of the stratum corneum are
9
16
16
16
16
associated with dry skin (Rawlings, 2003; Coderch et al., 2003;
Draelos, 2012). In practice, ceramide 1 (CAS no. 100403-19-8) or 3
(CAS no. 100403-19-8) applied individually or as a mixture in the
form of an emulsion synergistically improves the skin barrier
function in humans (Yilmaz and Borchert, 2006; Machado et al.,
2007; Huang and Chang, 2008). Furthermore, some ceramide- or
pseudoceramide-dominant emulsions can decrease the severity of
pruritus and trans-epidermal water loss (TEWL) in patients (Luk
et al., 2009; Kircik and Del Rosso, 2011). Recently, it was reported
that ceramides are also used as one of the major constituents of
topical formulations for rosacea (Sparavigna et al., 2014) and facial
atopic eczema (Miller et al., 2011; Puviani et al., 2014).
Despite these attributes, little is known about the possible
toxicity of ceramides. In particular, the toxicity data for exogenously
applied natural ceramides, such as ceramide 3, is very limited.
Recently, the Cosmetic Ingredient Review (CIR) Expert Panel
reviewed the safety of ceramides. They concluded that ceramide
ingredients are safe in the cosmetics in the present practices of use
and concentration based on the unpublished data submitted by
Personal Care Product Council (CIR, 2015). For pseudoceramide- or
Fig. 1. Chemical structure of ceramides and pseudo-ceramide. Phytosphingosine serves as a base for ceramide 3, which is amide-linked to a non-hydroxy fatty acid. Sphingosine and
6-hydroxysphingosine serve as a base for ceramide 2 (CAS no. 100403-19-8) and 8, respectively.
10
S.M. Choi, B.-M. Lee / Food and Chemical Toxicology 84 (2015) 8e17
Table 1
Information on ceramide 3.
CAS No.
EINECS No.
INCI name
IUPAC name
100403-19-8
309-560-3
Ceramide 3
Ceramide NP
e
Adopted from Gottschalck and Bailey (2010).
ceramide-dominant formulations, some systemic toxicity data are
available, but they can only be used as reference data for the direct
risk assessment of natural ceramides. Consequently, there is no
consensus concerning the safe concentration of ceramides used in
cosmetic or personal products worldwide.
This study was undertaken to review the toxicological characterization of ceramides and pseudo-ceramides, and to carry out risk
assessment for ceramide 3. The searches have been carried out in
the Medline/PubMed and TOXNET database until April 2015 using
predefined keywords such as ceramide(s), ceramide 3, ceramide
NP, and pseudo-ceramide(s). This review may help provide scientific rationale for the regulation of ceramide use in cosmetic
products.
2. Physical and chemical properties
Ceramides contain one of the three types of sphingoid bases:
sphingosine, phytosphingosine, or 6-hydroxysphingosine. They are
amide-linked to a non-hydroxy fatty acid (e.g., ceramide 2, 3, and
8), a-hydroxy fatty acid (ceramide 5, 6, and 7), or u-hydroxy acid
(ceramide 1 and 4) (Robson et al., 1994; Ponec et al., 2003). In
particular, ceramide 3 consists of a phytosphingosine backbone
acylated with a saturated fatty acid (stearic acid) (Fig. 1). The major
physicochemical properties are detailed in Table 1.
3. Production and usage
Endogenous ceramides are synthesized at the cytosolic side of
the endoplasmic reticulum through the action of several
sphingolipid-metabolizing enzymes (Novgorodov and Gudz, 2011).
For commercial mass production, ceramides can be obtained from
natural sources or they can be synthesized. Although synthetic
analogues of natural ceramides and pseudo-ceramides usually do
not have the optimal three-dimensional molecular configuration,
they are of commercial value owing to their equivalent absolute
molecular configuration to human skin ceramides (Lee et al., 2003).
Naturally derived ceramides are biologically similar to the ceramides found in the human skin. They are derived from animal tissues
or organs (mainly the sphingolipids found in the brain and spinal
cord) or are manufactured by yeast (Pacha and Hebert, 2012).
Table 2
Ceramide concentration in cosmetic products.
Product types
Concentration (%)
Lotion
Lip gloss
Lip balm
Lipstick
Eyeshadow
Essence
Cream
Cleansing gel
Cleansing cream
Powder
0.01e0.3
0.01e0.05
0.01
0.01e0.2
0.1
0.01e0.5
0.01e0.7
0.1
0.01
0.01
Data source: KCII (Foundation of Korea Cosmetic Industry
Institute), 2012.
Contamination with prions that can transmit bovine spongiform
encephalopathy is a concern when using animal sources (Adkin
et al., 2010). The other source of sphingolipids is the yeast Pichia
ciferrii, which differs from all the other known microbial sources
because it is able to produce large quantities of phytosphingosine in
the form of tetraacetyl phytosphingosine through fermentation
(Barenholz and Gatt, 1969, 1972; Barenholz et al., 1973). The stereochemical configuration of the extracellular sphingolipids produced by P. ciferrii is identical to that found in human skin
(Wickerham and Stodola, 1960). Advances in biotechnology have
enabled effective production of sphinganine and sphingosine for
cosmetic and pharmaceutical applications (Schorsch et al., 2009;
€rgel et al., 2012). Typically, fatty acids are coupled with phytosBo
phingosine for the commercial scale production of ceramides (Lee
et al., 2003; Wollenweber and Farwick, 2006). As reported by
Kwun et al. (2006), optimal ceramide production can be achieved
using another yeast Saccharomyces cerevisiae, when grown at pH
6.0 and 30 C.
Ceramide 3 is used in a wide range of cosmetic products,
including lipstick, body lotion, face cream, shampoo, and facial
cleanser. In South Korea, a survey of the cosmetic products showed
that liquid cosmetics contain ceramide at concentrations of
0.01e0.3 g/100 g, and cream cosmetics contain 0.01e0.7 g/100 g
(Table 2) (KCII, 2012).
4. Human exposure
Several clinical studies on formulations containing pseudoceramide and ceramide have been published (Table 3). In one
case of ceramide-mediated allergic contact dermatitis, a 22-yearold female showed positive response in patch test to 0.25% and 0.5%
(w/w) concentrations of a chemically synthesized hydrophilized
ceramide resembling type 2 ceramide (Yajima, 2002). In another
study, the efficacy and safety of emulsions containing ceramide 1
and/or ceramide 3 were evaluated in 15 Asian women with healthy
skin (Huang and Chang, 2008). The participants applied the
emulsions twice daily for 28 days. A beneficial synergistic effect of
the ceramides applied as a combination emulsion was evident from
skin hydration and TEWL. A skin erythema test conducted in the
same study revealed that at 0.02% (w/w), both ceramides did not
induce any visual skin irritation during the 4-week study period.
Kircik and Del Rosso (2011) investigated EpiCeram™, a
ceramide-dominant triple lipid formulation containing a 3:1:1
molar ratio of a pseudo-ceramide (ceramide N-(2-hydroxyethyl)-2penta-decanolylhexadecanamide), cholesterol, and free fatty acid
(Pediapharm, Ile des Sœurs, Quebec, Canada). The ceramidedominant formula was applied twice daily for 3 weeks to 65 subjects with atopic dermatitis (3 monthse16 years of age; mean age:
7.4 years). Three of the 65 subjects showed treatment-related
adverse but non-serious events that included mild pain, irritation,
and pruritus.
In a separate study conducted by Lowe et al. (2012), the same
formula was applied for 6 weeks to 10 infants (0e4 weeks of age)
with a family history of allergy. Adverse events including heat rash,
facial eczema, folliculitis, dry skin, bronchiolitis, cough/fever,
conjunctivitis, reflux, and common cold were observed. However,
since all of these events are common conditions in infancy, it could
not be concluded that they arose solely in response to the
formulation.
Another clinical study evaluated Curel® (Kao, Tokyo, Japan), a
commercially available moisturizing cream containing pseudoceramide (Luk et al., 2009). The cream was applied to subjects
with senile xerosis twice daily for 3 weeks. Significant improvements in skin hydration and symptom scores were evident. The
exact formulation of the pseudo-ceramide and its concentration in
S.M. Choi, B.-M. Lee / Food and Chemical Toxicology 84 (2015) 8e17
11
Table 3
Clinical study results of ceramides and pseudo-ceramides.
Test article
Subject
Synthesized hydrophilized
ceramide
Ceramide 1, ceramide 3 or the
mixture containing emulsion
EpiCeram™ (Pseudo-ceramidedominant triple lipid formula)
Allergic contact dermatitis 0.25, 0.5%
patients
Healthy volunteers
0.02%
EpiCeram™ (pseudo-ceramidedominant triple lipid formula)
Infant (0e4 weeks of age)
with a family history of
allergic disease
Senile xerosis patients
Atopic dermatitis patients
(mean aged 7.4 years)
Curel® (pseudo-ceramide
containing moisture cream)
Curel® (pseudo-ceramide containing Atopic eczema patients
moisture cream)
(5e18 years old)
Concentration
Duration
Results
Ref.
e
Positive response
Yajima, 2002
28 days
No skin irritation (both
single and the mixture)
Mild pain, irritation and
pruritus (3/65 patients)
Huang and Chang, 2008
Kircik and Del Rosso, 2011
3:1:1 molar ratio of pseudo- 3 wks
ceramide, cholesterols, free
fatty acid
No treatment related AEs Lowe et al., 2012
3:1:1 molar ratio of pseudo- 6 weeks
ceramide, cholesterols, free
fatty acid
Not described
3 weeks, twice No treatment related AEs Luk et al., 2009
a day
8%
4 weeks, bid
No safety data
Hon et al., 2011
information given
AE: adverse effect.
the applied cream remains proprietary. No adverse events occurred
during the study period, and the moisture cream was well tolerated
by the subjects. A similar study was subsequently conducted with
33 subjects aged 5- to 18-years-old who had atopic eczema (Hon
et al., 2011). A cream containing 8% synthetic pseudo-ceramide
was applied to the flexures and areas affected with eczema twice
daily for 4 weeks. Although skin hydration improved significantly,
no deterioration in TEWL or eczema severity was observed.
Fig. 2. Metabolic pathway of ceramide. The balance between generation and degradation of various enzymes regulate the intracellular levels of ceramide. Abbreviations: SMases,
Sphingomyelinases; SM, sphingomyelin; SMS, sphingomyelin synthase; Sph, sphingosine; SK, sphingosine kinase; GCS, glucosylceramide synthase; GC, glucosylceramide; S1P,
sphingosine 1-phosphate; S1PP, sphingosine 1-phosphate phosphatase; C1P, ceramide-1-phosphate; CDase, ceramidase; C1PP, ceramide-1-phosphate phosphatase; CK, ceramide
kinase; CRS, cerebrosidase; CS, ceramide synthase.
Adopted and modified from Pettus et al. (2002) and Tani et al. (2007).
12
S.M. Choi, B.-M. Lee / Food and Chemical Toxicology 84 (2015) 8e17
5. Pharmacokinetics
5.1. Absorption
Oral absorption of ceramide was evaluated in Hairless Wistar
Yagi (HWY) rats after single administration of 3H-ceramide at a
dose of 30 mg/kg. The Cmax was 2.75 ng eq./ml at 10.67 h and
decreased with a T1/2 of 67.12 h. The area under the curve AUC0-144
and AUC0-inf were estimated as 211.74 and 287 ng eq. h/ml,
respectively (Ueda et al., 2009).
Synthetic pseudo-ceramide SLE66 (CAS no. 110483-07-3),
which has a molecular structure analogous to the natural nonhydroxy fatty acid ceramide, has been developed by the Kao
Corporation (Tokyo, Japan) for patients with atopic dermatitis. The
absorption of [14C]-SLE66 was evaluated following oral and
dermal administration to SpragueeDawley (SD) rats (Morita et al.,
2009a). Oral administration at dose level of 1000 mg/kg resulted
in detectable blood concentrations from 30 min to 72 h. The Tmax
and Cmax were estimated to be 4 h and 39.77 mg eq./ml, respectively. In addition, the AUC(0-72) and AUC(0-inf) were calculated as
945 and 1080 mg eq. h/ml, respectively. A dermal pharmacokinetic study was also conducted using [14C]-SLE66 at a dose of
1000 mg/day or as a formulation (8%) at dose level of 160 mg/kg
to normal and abraded skin of SD rats. Application of [14C]-SLE66
to normal skin did not produce systemic absorption at any time
point. However, when applied to abraded skin as a formulation, a
small quantity of radioactivity was detected after 2e8 h (Tmax,
6 h). The Cmax was estimated to be 0.28 mg eq./ml, which is 0.7% of
oral Cmax. Based on these results, the authors concluded that this
pseudo-ceramide is unlikely to be absorbed following application
to normal skin.
5.2. Distribution
Ueda et al. (2009) reported that the high concentration of
radioactivity was observed in the adrenal gland, liver, mesenteric
lymph node, and small intestine at 12 and 24 h after oral administration of 3H-ceramide to HWY rats at a dose of 30 mg/kg. The
radioactivity also detected in the skin, which reached 4-fold as high
as the blood concentration at 120 h after administration.
5.3. Metabolism
Morita et al. (2009a) reported that the aforementioned pseudoceramide, SLE66, is primarily absorbed unchanged in plasma at 4 h
following oral administration. The remaining amount of the parent
compound found in plasmadnearly 59%dwas metabolized to
multiple unidentified minor metabolites. The endogenous level of
ceramide is controlled by the activities of various enzymes. Ceramide can be generated and eliminated through multiple routes
(Pettus et al., 2002). Among these metabolic pathways, a series of
ceramide/sphingosine/sphingosine-1-phosphates are known to
function not only as intracellular second messengers, but also as
extracellular messengers (Tani et al., 2007). As shown in Fig. 2,
ceramide is produced when sphingomyelinases cleave sphingomyelin. Ceramide is then deacylated to sphingosine by ceramidase.
Sphingosine is phosphorylated to sphingosine 1-phosphate by
sphingosine kinase. Sphingosine 1-phosphate is rapidly degraded
into either hexadecenal or phosphoethanolamine and converted to
phosphatidylethanolamine and palmitate, respectively (Cuvillier,
2002; Spiegel and Kolesnick, 2002).
5.4. Excretion
Excretion of
3
H-ceramide was investigated after single oral
administration to HWY rat at a dose of 30 mg/kg. The radioactivity
recovered within 96 h was 87.4%, 4.8%, and 0.6% in feces, urine, and
expired air, respectively (Ueda et al., 2009).
Excretion of SLE66 was investigated using [14C]-SLE66 following
oral administration (1000 mg/kg) to SD rats (Morita et al., 2009a).
The excretion of radioactivity for up to 24 h in urine, feces, and
expired air was 5.7%, 84.8%, and 0.6%, respectively. It was concluded
that the main excretion route of pseudo-ceramide is via the feces.
When administered orally, a small amount is converted to 14CO2.
6. Toxicological effects
6.1. General toxicity
Ceramide is a lipid-signaling molecule that is involved in various
cellular events, including proliferation, differentiation, and
apoptosis (Hannun, 1994; Kolesnick and Kronke, 1998; Hannun and
Obeid, 2002). Because these endogenous ceramides are found at
trace levels in living organisms, they pose no safety concern.
However, safety should be monitored when using highly functional
synthetic pseudo-ceramides.
Morita et al. (2009b) evaluated the potential acute adverse effects of SLE66 in rats. Although several clinical signs, including
piloerection, hunched posture, and pallor of the extremities, were
observed in the oral toxicity study, all these signs resolved within 2
days following administration. Furthermore, no clinical signs were
observed with dermal application of SLE66. The LD50 values of this
pseudo-ceramide were estimated to be greater than 5 g/kg and 2 g/
kg when administered orally and dermally, respectively. Another
synthetic pseudo-ceramide, Bio-391 [N-(2-hydroxyethyl)-2pentadecanolyl-hexadecanamide; CAS no. 791838-11-4], was also
evaluated for acute toxicity according to the Organization for Economic Co-operation & Development (OECD) guidelines, although
detailed information was not available (Uchida et al., 2008). The
LD50 value exceeded 2000 mg/kg in rodents, indicating that serious
acute toxicity was unlikely.
A 4-week repeated toxicity study of pseudo-ceramide SLE66
was carried out in SD rats administered SLE66 via gavage once daily
for 28 days at doses of 0, 150, 400, or 1000 mg/kg (Morita et al.,
2009b). In a separate study, SLE66 was also applied daily for 5
days/week for 28 days at doses of 0, 100, 300, or
1000 mg kg1 day1 to the dorsal clipped area of rats (Morita et al.,
2009b). In both studies, no mortality, abnormal clinical observations, or changes in body weight or food consumption were
observed. Furthermore, there were no dose-related changes in
hematological parameters, serum chemistry, organ weight, or histopathological indicators. Based on these results, the no-observedadverse-effect level (NOAEL) for systemic toxicity following oral or
dermal administration of SLE66 was estimated to be 1000 mg/kg/
day.
6.2. Genotoxicity
No mutagenic potential was reported in the Ames test in the
presence and absence of S9 mixture (Morganti and Fabrizi, 1999).
The mutagenicity of SLE66 was also evaluated using the Ames and
chromosomal aberration tests (Morita et al., 2009a). In the Ames
test, no significant or dose-responsive increase in the mean number
of revertant colonies was recorded in the presence or absence of S9
metabolic activation. The chromosomal aberration test revealed a
lack of dose-related increase in the number of cells at metaphase in
human lymphocytes exposed to SLE66. These results suggest that
SLE66 is neither clastogenic nor mutagenic.
S.M. Choi, B.-M. Lee / Food and Chemical Toxicology 84 (2015) 8e17
13
6.3. Carcinogenicity
6.7. Ocular toxicity
Similar to genotoxicity, no reports on the carcinogenicity of
ceramides and pseudo-ceramides have been published.
However, it is well studied that the endogenous ceramides have
tumor suppressing effects through various mechanism such as
apoptosis, necrosis and autophage (Dany and Ogretmen, 2015).
Furthermore, the levels of ceramides are up-regulated when cancer
cells are exposed to anticancer drugs and radiation (Ponnusamy
et al., 2010) and defects in ceramide generation and metabolism
in cancer cell reduced the response to chemotherapy (Galadari
et al., 2015). For example, Koybasi et al. (2004) reported that C18
ceramide, a product of ceramide synthase 1, was significantly lower
in the majority of tumor tissues of patients with head and neck
squamous cell carcinoma (HNSCC) than that in noncancerous
adjacent head and neck tissues. A further study demonstrated that,
the decreased C18 ceramide level was significantly correlated with
lymphovascular invasion and nodal metastasis in patients with
HNSCC (Karahatay et al., 2007). The expression of ceramide was
also investigated in 23 patients with leukoplakia larynx (Yuan et al.,
2007). The ceramide expression levels decreased gradually from
normal tissue via precancerous lesions to laryngeal carcinoma,
which suggests a potential tumor suppressive effect of ceramide.
Morganti and Fabrizi (1999) reported that ceramide 3 induced
light irritation when instilled into (27 mg) the eye of three rabbits.
However, the detailed experimental condition and result were not
described in the study.
An eye irritation study was conducted for SLE66 (Morita et al.,
2009b). In this study, 70 mg (0.1 ml) of SLE66 was applied to the
lower everted eyelids of NZW rabbits and 100 mg of SLE66 was
instilled into the eyes of Japanese white. There was no corneal
damage or iridial inflammation following SLE66 instillation,
although some enlargement of the conjunctival blood vessels was
observed in all animals 1 h after application. These changes
resolved 1e3 d after the instillation of SLE66. In a second set of
experiments, 100 mg SLE66 resulted in a mild conjunctival reaction
1, 3, and 6 h post-instillation according to the Draize score. These
changes were also restored to normal 24 h post-instillation.
6.4. Immunotoxicity
There have been no reports on the immunotoxicity of ceramides
or pseudo-ceramide.
6.5. Developmental/reproductive toxicity
Developmental toxicity for pseudo-ceramide SLE66 was
assessed in mated female Crl:CD(SD)IGS BR rats (25 heads/group)
(Morita et al., 2009c). The rats were administered SLE66 orally once
daily from gestation day 6 through day 19 (0, 150, 400, or
1000 mg kg1 day1). There were no changes in body weight,
gravid uterine weight, feed consumption, intrauterine growth, fetal
survival, or fetal malformations or developmental variations. Based
on these results, it was concluded that the NOAEL for developmental or maternal toxicity was 1000 mg kg1 day1.
6.6. Local toxicity
No non-clinical study has examined the local toxicity of
ceramides except for one brief study reported by Morganti and
Fabrizi (1999). In this study, ceramide 3 was not irritating after
application to the intact skin of three rabbits at the dose of 0.5 g.
However, adverse effects of the pseudo-ceramide SLE66, such as
irritation and pruritus, have been reported in clinical study, as
described in the section on human exposure data. In a preclinical
study, two independent investigations of SLE66 were conducted by
Morita et al. (2009b). For the skin irritation investigation, the
dermal application of SLE66 (0.5 g) did not induce any irritating
reaction or toxic signs in New Zealand White (NZW) rabbits. In a
skin sensitization study using guinea pigs, SLE66 did not produce
any toxic signs or dermal reactions following intradermal injection
(1% SLE55) or topical application (60% SLE66). Similar results were
observed following 20 applications over a 5-week period at a
concentration of 5% or 50% in human volunteers. In this human
study, no skin reaction was evident for either a single patch test
(0.02 ml of 5% SLE66) or a cumulative skin irritation test (5% and
50% SLE66 applied 20 times over 5 weeks) (Morita et al., 2009b).
6.8. Other toxicities
In a phototoxicity study, SLE66 was applied to the back of female
Hartley guinea pigs at a concentration of 0%, 5%, and 50% (Morita
et al., 2009b). There were no skin reactions regardless of ultraviolet exposure at any time point for any experimental group, except
for the positive control group (0.03% 8-methoxypsolaren). Morita
et al. (2009a) conducted a cytotoxicity test for SLE66 using a
three-dimensional human skin model (cornified normal human
epidermal cells). The cell viabilities of 20%~56% of SLE66 ranged
from 75% to 82%, whereas that of the vehicle was 87%. When an 8%
SLE66 medicated cream was applied, the cell viability was 103%
compared to the control. Therefore, it was concluded that SLE66 did
not produce obvious cytotoxicity.
7. Regulation status
Ceramide 3 is not classified as a restricted ingredient for cosmetics by the United States (FDA, 2015), Canada (Health Canada,
2014), member countries of the European Economic Union (EC,
2013), or South Korea (KFDA, 2015).
8. Risk assessment
There have been no scientific or systemic risk assessments for
ceramides owing to a lack of toxicological data that would enable
identification of NOAEL. Nonetheless, there are several reasons why
a risk assessment is needed.
First, although natural-source ceramides are not commonly
used for commercial purposes, they might pose a risk of contamination when they are derived from bovine central nervous systems.
Second, there is limited information on the toxicity of both
endogenous and synthetic pseudo-ceramides. Since ceramide is
Table 4
In-silico prediction of physico-chemical and ADME related properties.
Parameters
Ceramide 3
SLE66
Solubility
LogP
LogSw
Bioavailability (%)
AUCpo0-inf (mg/ml)
AUCiv0-inf (mg/ml)
Caco-2 permeability (106 m/s)
Highly insoluble
15.18
9.94
0
0
14.22
0
Highly insoluble
13.41
7.84
0
0
15.07
0
Abbreviations: ADME, absorption, distribution, metabolism, and excretion; AUCpo,
area under the curve per os; AUCiv, area under the curve intravenous; LogP, Log
octanol/water partition coefficient; LogSw, Log intrinsic water solubility.
14
S.M. Choi, B.-M. Lee / Food and Chemical Toxicology 84 (2015) 8e17
Table 5
Average application number of products per use day.
Product types
Body lotion
Number
Hands
Arms
Feet
Legs
Neck & throat
Back
Other partsa
Face cream
Lipstick
Shampoob
Facial cleanser (Lathering
and Non-lathering)b
a
b
Table 8
Calculated average SED based on the assumption that the various concentrations of
ceramide 3 are contained in final products (mg kg1 day1).
Ref.
Min.
Mean
Max.
e
e
e
e
e
e
e
e
e
1.00
1.0
2.12
1.52
0.95
1.11
0.43
0.26
0.40
1.77
2.35
1.11
1.6
e
e
e
e
e
e
e
e
e
2.14
3.2
Loretz et al., 2005
Body lotion
Loretz et al., 2006
Loretz et al., 2008
Face cream
Lipstick
Shampoo
Facial cleanser
Includes torso, face, hips, buttocks, and elbows.
The ratio of the number of applications to the number of use days.
Table 6
Average amount of product applied per application.
Product types
Body lotion
Face cream
Lipstick
Shampoo
Facial cleanser
Amount (g)
Lathering
Non-lathering
Ref.
Min.
Mean
Max.
0.05
0.00
0.000
67.89
0.33
0.57
4.42
1.22
0.010
11.76
2.56
2.58
36.31
21.01
0.214
0.39
10.67
14.61
Loretz et al., 2005
Loretz et al., 2006
Loretz et al., 2008
Data are the ratio of the total amount used to the total number of applications.
involved in various cellular processes as a second messenger, the
possibility of a disturbance in this elaborate regulation by exogenous ceramides or pseudo-ceramides should be considered carefully. Third, there is no standard concentration limits for the use of
ceramides, including pseudo-ceramide, as an ingredient in beauty
products.
8.1. Hazard identification and doseeresponse assessment
Because of the absence of an NOAEL value for ceramide 3, the
toxicity study results on pseudo-ceramide were used instead of
ceramide 3. Structure-based similarity approaches using three
diverse similarity techniques (Tanimoto, Dice, Cosine) have been
Table 7
Average amount of cosmetic or personal product applied per day (mg kg1 day1).
Product types
Body lotion
Face cream
Lipstick
Shampoo
Facial cleanser
Aggregate exposurea
Amount(mg kg
Hands
Arms
Feet
Legs
Neck & Throat
Back
Other parts
Lathering
Non-lathering
SED (mg kg1 day1)
Product types
1
day
1
)
Min.
Mean
Max.
1.77
1.27
0.79
0.93
0.36
0.22
0.33
0.00
0.00
7.22
8.80
15.20
28.09
156.17
111.97
69.98
81.77
31.68
19.15
29.47
35.99
0.39
217.56
68.27
68.80
822.93
1282.95
919.85
574.91
671.74
260.22
157.34
242.07
619.80
8.38
1255.97
284.53
389.60
6382.83
Data are calculated as average number of applications per use day average amount
of product applied per application. The mean body weight of 60 kg was assumed for
the adults.
a
In the case of facial cleanser, only non-lathering type was used for calculation.
Hands
Arms
Feet
Legs
Neck & Throat
Back
Other parts
Lathering
Non-lathering
0.5%
1%
3%
5%
10%
0.78
0.56
0.35
0.41
0.16
0.10
0.15
0.18
0.00
1.09
0.34
0.34
1.56
1.12
0.70
0.82
0.32
0.19
0.29
0.36
0.00
2.18
0.68
0.69
4.69
3.36
2.10
2.45
0.95
0.57
0.88
1.08
0.01
6.53
2.05
2.06
7.81
5.60
3.50
4.09
1.58
0.96
1.47
1.80
0.02
10.88
3.41
3.44
15.62
11.20
7.00
8.18
3.17
1.92
2.95
3.60
0.04
21.76
6.83
6.88
conducted. It has been reported that these indices have high predictive ability for similarity calculations (Bajusz et al., 2015). The
calculated values between ceramide 3 and pseudo-ceramide,
SLE66, using Discovery Studio v4.0 (Accelrys Software Inc.) were
0.940, 0.969, and 0.988 in Tanimoto, Dice, and Cosine coefficients,
respectively. These values demonstrate that ceramide 3 and SLE66
are highly similar in chemical structure. The physico-chemical and
pharmacokinetic properties were also predicted using an in silico
prediction tool. The descriptors of compounds, i.e. logP (octanol/
water partition coefficient, estimated by Kowwin), solubility, logSw
(intrinsic water solubility, estimated by Wskowwin), bioavailability, AUC, and permeability were determined using ACD Percepta
(ACD/Labs software). As shown in Table 4, it is predicted that the
two compounds have similar physico-chemical properties, which
predict similar toxicokinetics. The in silico prediction supports that
the toxicological profile of SLE66 can be applied to the risk
assessment of ceramide 3.
SLE66 (a long acyl chain ceramide analogous to natural ceramides) was applied to the dorsal clipped regions of male and female
rats at doses of 100, 300, or 1000 mg kg1 day1 once daily for 4
weeks (Morita et al., 2009b). This 4-week dermal application did
not result in any deaths, and the clinical observations were similar
in the vehicle control and the pseudo-ceramide treated group. No
body weight or food consumption changes or blood-related indications of toxicity were observed in any experimental group. At
terminal necropsy, no toxicologically meaningful changes were
seen either macroscopically or microscopically. Therefore, the
Table 9
The MOS of various cosmetic or personal products based on assumption that various
concentrations of ceramide 3 are contained in final product.
MOSa
Product types
Body lotion
Face cream
Lipstick
Shampoo
Facial cleanser
Hands
Arms
Feet
Legs
Neck & Throat
Back
Other parts
Lathering
Non-lathering
0.5%
1%
3%
5%
10%
206
288
460
394
1017
1681
1093
895
82,213
148
472
468
103
144
230
197
508
841
546
447
41,106
74
236
234
34
48
77
66
169
280
182
149
13,702
25
79
78
21
29
46
39
102
168
109
89
8221
15
47
47
10
14
23
20
51
84
55
45
4111
7
24
23
a
Calculated using the equation MOS ¼ NOAEL/SED, where NOAEL is 161 mg/kg
which is estimated in 8.1. “Hazard identification and dose response assessment”
section, and SED is the calculated value in Table 8.
S.M. Choi, B.-M. Lee / Food and Chemical Toxicology 84 (2015) 8e17
15
Table 10
The MOS of various cosmetic products produced in South Korea.
Lotion (Body)
Cream (Face)
Cream (hand)
Eyeshadow
Lipstick
a
b
c
Concentration (%)a
Estimated daily exposure (mg kg1 day1)b
SED
MOSc
0.01e0.3
0.01e0.7
0.01e0.7
0.1
0.01e0.2
123.20
24.14
32.70
0.33
0.90
0.0123e0.3696
0.0024e0.1690
0.0033e0.2289
0.0003
0.0001e0.0018
436e13,068
953e66,694
703e49,235
487,879
89,444e1,788,889
Data from KCII (Foundation of Korea Cosmetic Industry Institute), 2012.
Data from Hall et al., 2007, 2011; SCCS, 2012.
Calculated using NOAEL (161 mg/kg) in 8.1. “Hazard identification and dose response assessment” section.
NOAEL of SLE66 in rats was estimated to be 1000 mg kg1 day1.
For the margin of safety (MOS) approach, NOAEL can be converted
to a human-equivalent dose (HED) using the following equation:
SED mg kg1 day1 ¼ A mg kg1 day1 Cð%Þ=100
DAp ð%Þ 100
HEDðmg=kgÞ ¼ NOAEL 1000 mg=kgðrat; dermalÞ÷6:2a
¼ 161 mg=kg
a
Assumes 60 kg human. Conversion of animal doses to HED
based on body surface area (US FDA, 2005).
8.2. Exposure assessment
To conduct risk assessment of cosmetic products, exposure information was used for cosmetic and personal care products (Loretz
et al., 2005, 2006, 2008). In these studies, female subjects were
recruited from across the United States and were asked to record
their use of each product in a diary for 2 weeks. The average
number of product applications and the average amount per use
are shown in Tables 5 and 6. Total survey estimates were derived by
assuming that the 14 person-days contributed by each of the participants represented independent person-days of observations.
Therefore, the estimates were based on 4312 (14 person-days for
each of the 308 participants), 4200 (14 person-days for each of the
300 participants), and 4354 person-days (14 person-days for each
of the 311 participants) for body lotion, face cream, and lipstick,
respectively.
Based on the data in Tables 5 and 6, the average amount of
product applied each day was calculated and summarized in
Table 7. In the case of body lotion, it was assumed that the average
amount per application was equal to the amount used on all body
parts. Because there are no specific data regarding the dermal absorption of ceramide 3, calculation of the systemic exposure dosage
(SED) was conducted using the formula shown below. Regarding
dermal absorption, we assumed that 100% of ceramide 3 would
penetrate into the skin due to a lack of relevant data and assuming
Table 11
Aggregate exposure based SED and MOS assuming that various concentrations of
ceramide 3 are contained in the final products.
Ceramide 3 concentration
0.5%
1%
3%
5%
10%
SED (mg kg1 day1)
MOSa
Min.
0.14
0.28
0.84
1.40
2.81
b
the worst-case scenario (KFDA, 2011). The SED was also calculated
at various concentrations to suggest an upper limit value using the
MOS approach (Table 8).
Mean
Max.
Min.
Mean
Max.
4.11
8.23
24.69
41.15
82.29
31.91
63.83
191.48
319.14
638.28
1150
575
191
115
57
39
19
6
3
1
5
2
0
0
0
The values were calculated based on the aggregation of exposure to cosmetic and/or
personal products as shown in Table 7.
a
MOS ¼ NOAEL (161 mg/kg)/SED as described in the sections of 8.1e8.3.
b
Aggregate exposure (Min. amount, 28.09 mg kg1 day1) 0.5%/100 100%/
100.
The value A is the estimated daily exposure to the product as
calculated in Table 7, C (%) is the concentration of the ingredient in
the finished cosmetic or personal product on the application site,
and DAp (%) is the dermal absorption expressed as a percentage of
the test dose assumed to be applied in real-life conditions.
8.3. Risk characterization
The MOS values for various cosmetic and/or personal products,
which may contain ceramide 3, are summarized in Table 9. We
assume that ceramide 3 exists in these types of products at a wide
range of concentrations. There are no limitations on its usage at
present. The MOS's for all products exceeded 100 except for
shampoo, assuming that ceramide 3 is contained at a concentration
of 1% in the final product. Therefore, it can be concluded that if
ceramides are used at concentrations of within 1% in the final
products, there would be no safety concerns. These analytic results
are consistent with a previous report of Huang and Chang (2008).
As described in the “Human Exposure Data” section, ceramide 1,
ceramide 3, or emulsions containing both were all safe when
applied twice daily over a period of 28 days. In this study, each
volunteer applied 0.2 g of the ceramide emulsions to each forearm.
Based on the 0.02% of ceramide 3 contained in this emulsion, the
SED can be calculated as 0.0013 mg kg1 day1 (200 mg 2
forearm$601 kg-1 day1 0.0002). In this case, the MOS would be
higher than 100,000, which indicates no risk for humans.
For body lotion, the MOS values varied depending on the
application sites. The application site resulting in the lowest MOS
was the hands, while the back was the least sensitive. However,
considering that the most common application sites for body lotion
are the arms and legs, the reasonable concentration limit of ceramide 3 in body lotions should be less than 1%. Regardless of
lathering, it is also reasonable that facial cleansers should include
less than 1% of ceramides in the final product. However, face creams
containing up to 3% ceramide concentrations in the final products
do not pose health risks. The only cosmetic product with no predicted risk up to 10% ceramide concentration in the final product
was lipstick. Even if the concentration of ceramide 3 used in a
lipstick product is 10%, the MOS would still be greater than 4000.
At present, the most critical point for the risk assessment of
ceramide 3 is the lack of systemic and relevant toxicity data. For a
more accurate risk profile prediction for ceramide 3, detailed
toxicity studies are needed. Considering this fundamental limitation, a conservative approach should be used for risk assessment.
From this perspective, it can be concluded that the concentration of
16
S.M. Choi, B.-M. Lee / Food and Chemical Toxicology 84 (2015) 8e17
ceramides in most types of cosmetics or personal products except
for shampoos, face creams, and lipsticks, should not exceed 1%.
In practice, the concentrations of ceramide 3 in several cosmetic
products produced in South Korea range from 0.01% to 0.7%. Based
on previous reports for estimated daily exposure data (Hall et al.,
2007, 2011), the calculated MOS values were above 100 regardless of cosmetic type (Table 10). Therefore, the use of ceramide 3 in
consumer products at concentrations lower than 1% would pose no
safety concerns, but the upper limit of safety may vary depending
on the product.
The possibility of aggregate exposure is also important considering factor in chemical safety assessment (Kennedy et al., 2015a,
2015b). For this purpose, a tiered approach is recommended
which is consisted of a conservative tier 1 for the screening level
assessment and more complex tier 2 for realistic aggregated
exposure modeling (Dudzina T et al., 2015; Tozer SA et al., 2015).
However, no standard methods have been adopted until now. In
practice, the Scientific Committee on Consumer Safety (SCCS) used
global daily exposure value when estimate the aggregate exposure
of preservatives assuming the worst-case scenario (SCCS, 2012).
Therefore, we have conducted rough deterministic estimation, that
is, the daily exposures of all product types were added. In the case
of facial cleanser, non-lathering type was used for calculation
instead of lathering type for considering the worst-case scenario. As
a result, the MOS value was only 39 when assumed that all products
contain as low as 0.5% ceramide 3 and used average daily amount
(Table 11). However, even though it is assumed that all products
contain 5% ceramide 3, the MOS values exceed 100 when minimal
amounts of ceramide 3 are used (Table 11).
9. Summary and conclusion
Although ceramide 3 is widely used in personal and cosmetic
products, data on its specific concentration limits are lacking. We
performed a risk assessment for ceramide 3 in various types of
personal or cosmetic products, under the assumption that these
products contained 0.5e10% ceramide 3 and the rate of dermal
absorption was 100%. The MOS 100, which indicates no risk (Kim
et al., 2013; Kang et al., 2014; Chen et al., 2014), varied depending
on product type and application site. Nevertheless, it can be
concluded that there may be sufficient margin of safety for systemic
toxicity to humans if ceramide 3 is used less than 1% as an ingredient for cosmetic products. For a more accurate prediction of the
human health risk, however, appropriate toxicity studies for ceramide 3 and the other ceramides will be required in the future.
Conflict of interest
This work was supported by a grant from the Foundation of
Korea Cosmetics Industry Institute in 2013 and by a grant
(14172MFDS975) from Ministry of Food & Drug Safety in 2014.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.fct.2015.07.012.
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