Toxicology Letters 294 (2018) 205–211

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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet

Mini review

Critical evaluation of 2-ethylhexyl acrylate dermal carcinogenicity studies T

using contemporary criteria
⁎
Sandra Murphya, Robert Ellis-Hutchingsb, , Lavorgie Finchc, Stefanie Welzd, Karin Wienchd
a
Kennett Square, PA, 19348, United States
b
The Dow Chemical Company, Midland, MI, 48674, United States
c
Arkema Inc., King of Prussia, PA, 19406, United States
d
BASF SE, Ludwigshafen am Rhein, Germany

A R T I C LE I N FO A B S T R A C T

Keywords: Skin tumors have been observed in C3H/HeJ mice following treatment with high and strongly irritating con-
2-Ethylhexyl acrylate centrations of 2-ethylhexyl acrylate (2-EHA). Dermal carcinogenicity studies performed with 2-EHA are re-
Nongenotoxic irritant viewed, contrasting the results in two mouse strains (C3H/HeJ and NMRI) under different dosing regimens.
Dermal carcinogenesis Application of contemporary evaluation criteria to the existing dermal carcinogenicity dataset demonstrates that
C3H/HeJ mouse
2-EHA induces skin tumors only at concentrations exceeding an maximum tolerated dose (MTD) and in the
Maximum tolerated dose
Strain selection
immune-dysregulated C3H/HeJ mouse model. Overall, the available chronic toxicity and genotoxicity data on 2-
EHA support a non-genotoxic chemical irritant mechanism, whereby chronic irritation leads to inflammation,
tissue injury, and wound repair, the latter of which is disrupted in C3H/HeJ mice and leads to tumor formation.
Tumor response information in excess of an MTD should not be considered in a human hazard or risk assessment
paradigm. For the purposes of an appropriate hazard assessment, 2-EHA did not cause or initiate dermal car-
cinogenesis in an immune competent (NMRI) mouse model, and, even in the immune compromised C3H/HeJ
model, did not induce skin tumors at doses which did not exceed the MTD.

1. Introduction ceptions occurring at the site of initial contact. Rodent forestomach

2-Ethylhexyl acrylate (2-EHA) is a reactive monomer ester. It is
produced, distributed, stored and consumed in closed systems. 2-EHA is
used in the production of coatings and inks, adhesives, sealants, plastics
and elastomers. Occupational exposure can occur during maintenance,
sampling, testing, manual transfer, or other procedures. Safety Data
Sheets and technical information bulletins provide information on
personal protective equipment and industrial hygiene measures that are
appropriate to control dermal or inhalation exposures. This substance is
not sold for direct consumer use, but it is used as a raw material to make
polymers for a variety of goods used by consumers or construction
personnel and could be present in trace amounts as residual monomer
in consumer products (BAMM, 2014).
Suh et al. (2018) recently reviewed the available data addressing
genotoxicity, mutagenicity, and carcinogenicity of 2-EHA and related
short chain esters of acrylic acid which have been tested in one or more
oral, dermal, or inhalation studies using various animal models. These
materials did not show any increase in tumor incidence, with two ex-
tumors were induced by oral gavage dosing with ethyl acrylate (EA), for
which Thompson et al. (2018) recently assessed the mode of action and
Proctor et al. (2018) described an adverse outcome pathway. The other
exception to a lack of tumor induction with short chain acrylate esters is
skin tumors observed following dermal application of 2-EHA to C3H/
HeJ mice. Both the forestomach and skin cancers occurred only after
chronic exposure to high doses and long-term tissue damage, which has
been shown to be preventable in stop-dose recovery studies (Suh et al.,
2018).
In contrast to EA, the mechanism for 2-EHA-induced tumorigenesis
has not yet been extensively assessed. As will be examined further in
this paper, chronic dermal application of high and strongly irritating
concentrations of 2-EHA caused skin tumors in C3H/HeJ, but not NMRI
mice (DePass et al., 1984; Mellert et al., 1994; Wenzel-Hartung et al.,
1989). Chronic irritation leading to inflammation and tissue injury is
well recognized to contribute to cell replication and tumor progression
(Abel et al., 2009; Mueller, 2006; Singh et al., 2014). This paper dis-
cusses key factors likely contributing to the differential response in

⁎
Corresponding author at: The Dow Chemical Company, Toxicology and Environmental Research Consulting (TERC), 1803 Building, Midland, MI, 48674, United States.
E-mail address: REllis-hutchings@dow.comc (R. Ellis-Hutchings).

https://doi.org/10.1016/j.toxlet.2018.05.016
Received 18 April 2018; Received in revised form 10 May 2018; Accepted 11 May 2018
Available online 15 May 2018
0378-4274/ © 2018 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
S. Murphy et al.
mice, particularly the doses utilized in these studies and the unique
background biology of the C3H/HeJ mouse strain.
2. Mice as a test system in research and regulatory testing
Scientists and regulatory authorities have generally employed ro-
dent bioassays to evaluate potential carcinogenicity. Due to their lim-
ited life span and widespread use in experimental studies a large
amount of information has accumulated on rat and mouse physiology
and pathology. Mice have been used extensively to study the potential
for various biological, physical and chemical agents to produce skin
tumors (Abel et al., 2009). While the utility of mouse data in human
risk assessments has been questioned by some due to the high tumor
susceptibility of some strains (OECD, 2012), Megosh et al. (2002)
proposed that mouse models may be the only realistic approach for
identifying and eventually characterizing the functions of relevant
genes and as a method to aid in the development of preventive strate-
gies for those at increased risk of cancer due to their genetic profile.
Many skin painting studies conducted for use in chemical hazard as-
sessment were conducted with C3H/HeJ mice because of their low
spontaneous skin tumor incidence. As these studies were predominantly
conducted several decades ago their design lacked important criteria
integral to contemporary requirements.
Current guidelines for design and conduct of carcinogenicity studies
include criteria to ensure valid and reliable data are generated in these
animal studies (OECD, 2012). Aspects requiring consideration include:
selection of an administration route that is appropriate for the inherent
physical-chemical properties of the substance, conduct of well-designed
shorter duration preliminary toxicity studies to initially characterize the
response to treatment, and proper definition of the maximum tolerated
dose (MTD) to prevent overwhelming the normal physiological re-
sponses and confounding the effects caused by the test material. Fol-
lowing discussion of the physical-chemical properties and the available
study results, the data will be critically evaluated against these criteria
to characterize the carcinogenic hazard of 2-EHA.
3. 2-EHA Physical−chemical properties and toxicology data
3.1. Physical-chemical properties appropriate for dermal carcinogenesis
studies
Dermal administration in carcinogenicity studies is an appropriate
route if it is relevant to potential exposures in humans and if the phy-
sical and chemical characteristics of the test substance support it.
Table 1 shows physical-chemical and toxicological properties relevant
to dermal exposures for 2-EHA. Given the low vapor pressure of 2-EHA,
high skin absorption, and the limitation of potential human exposures
to non-oral conditions for this monomer (BAMM, 2014) the dermal
route is an appropriate administration route for animal studies for this
substance. As 2-EHA is a reactive monomer, it is not surprising that it is
a moderate skin irritant and a skin sensitizer. Point of contact effects are
important to consider in the dose selection for dermal carcinogenicity
studies and are typically examined as part of an appropriately designed
range-finding study.
Table 1
2-Ethylhexyl acrylate physical-chemical and toxicological properties relevant to dermal exposure.
CAS No. Molecular Weight Water Solubility (mg/L @ 25 °C) Log Pow (25 °C)
103-11-7 184.3 9.6 4.64
a
GastroPlus modeling of 1 mg/kg in a 70 kg 30 year old male.
b
Hazard classification of “H315: Causes skin irritation” based upon findings in acute irritation studies (BAMM, 2014).
Toxicology Letters 294 (2018) 205–211
3.2. Cancer screening study in C3H/HeJ mice
The first C3H/HeJ mouse dermal cancer study was a screening study
design and, in addition to 2-EHA, several other short chain alkyl ac-
rylate monomers were tested using this model (Table 2) (DePass et al.,
1984; Gordon et al., 1991). The test concentrations for these screening
studies were set based upon observed gross irritation in a 2-week range-
finding study, a pre-test which is considered inadequate in both dura-
tion of dosing and evaluation of the site of application in view of
contemporary criteria which will be discussed below. The limited de-
tails and lack of microscopic evaluation in these studies makes com-
parisons of skin condition at study termination difficult. In the skin
painting cancer screening studies the substances were applied, un-
diluted or as a solution in acetone, to the shaved skin of the in-
trascapular region for 5 days per week. The test site was not occluded,
so vapor pressure, water solubility, and lipid solubility are important
characteristics that influence the test substance contact time with the
skin surface. Less volatile, lipophilic materials like 2-EHA are able to
remain on the skin longer than more volatile materials like ethyl ac-
rylate.
3.3. Standard mouse carcinogenicity studies
The tumor response in the screening skin painting study in C3H/HeJ
mice was unexpected, and prompted two more standard scale studies
using C3H/HeJ and NMRI mice (Table 3). The outcome of the follow-up
studies varied by mouse strain. Groups of 80 C3H/HeJ mice received 3
applications per week of 21, 43 or 86.5% 2-EHA. Evaluation of the
application site after 12 weeks of treatment showed eschar in 67 (84%),
49 (61%) and 52 (65%) of mice administered 86.5%, 43% and 21% 2-
EHA, respectively, prompting the addition of a 2.5% group and termi-
nating treatment of the 43% groups after 6 months. At the end of the
study, eschar was noted in all groups in a dose related manner, and
scaling was also observed in the majority of animals receiving 21 or
86.5% 2-EHA. The previously observed increased tumor incidence was
confirmed in the C3H/HeJ study at chronic irritating doses (∼21% or
85%). In the 43% 2-EHA group where applications were stopped after 6
months, skin damage recovered after 7 weeks without treatment and no
skin tumors were observed. No skin tumors occurred in the control or
2.5% groups, the latter of which developed only transient early irrita-
tion and, at termination, exhibited only microscopic focal/minimal
eschar (BASF, 1986; Wenzel-Hartung et al., 1989) (See Table 3).
In the study using NMRI mice, chronic application and initiation-
promotion protocols were used. Groups of 80 males were exposed
dermally 3 times/week to 25 μl of 21.5, 43 or 85% 2-EHA, acetone, or
0.015% of benzo(a) pyrene (BaP) and split at 7 months. Half of each
group continued treatment until termination, the other half were un-
treated for two months then treated with 12-O-tetradecanoylphorbol-
l3-acetate (TPA) for 20 weeks, and untreated until termination. At 12
weeks and 24 months, hyperkeratosis, hyperplasia, crust formation and
ulceration were observed in a dose-related manner in the 2-EHA groups
(BASF, 1992). Hyperplasia, lymphocyte infiltration, increased macro-
phages and dermal fibrosis occurred but were not dose related
(Table 3). One squamous cell papilloma (SCP) was found in the acetone
negative control group. TPA promotion caused a SCP in one animal per
group. These tumors were attributed to TPA irritation. In the BaP po-
sitive control group, 2 SCP and 31 squamous cell carcinomas (SCC)
Vapor Pressure (hPa 25 °C) Predicted Skin Absorptiona Skin irritation Skin sensitizer
b
0.24 99.60% Moderate Yes
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S. Murphy et al. Toxicology Letters 294 (2018) 205–211

Table 2
Screening studies: C3H/HeJ mice dosed at 25 μl in acetone, 3 doses/week, lifetime exposure.
Substance Acrylic Acida Ethyl Acrylatea n-Butyl 2-Ethylhexyl iso-Octyl Acrylatec
Acrylatea Acrylateb

CAS # 79-10-7 140-88-5 141-32-2 103-11-7 29590-42-9

Concentration 1% 100% 1% 75% 5%
Carcinogenicity – – – + –
Reported Irritation at Necropsy NR epidermal necrosis, dermatitis, dermal NR NR crusting, dermatitis, hyperkeratosis, epidermal
fibrosis, hyperkeratosis hyperplasia and melanosis
Vapor Pressure (hPa 25 °C) 5.29 40 (21 °C) 5 (22 °C) 0.24 1 (20 °C)
Log Pow (25 °C) 0.46 1.18 2.38 4.64 4.5-4.7
Predicted skin absorptiond 68.0% 68.0% 99.3% 99.6% 99.6%

NR - Data on skin condition not reported at study termination.

a
DePass et al. (1984).
b
DePass et al. (1985).
c
Gordon et al. (1991).
d
GastroPlus modeling of 1 mg/kg in a 70 kg 30 year old male.

Table 3
Chronic dermal studies – 2-ethylhexyl acrylate, 3 doses/week, lifetime exposure.
Strain (Sex) n Conc. % Dose vol. (μL) mg/kg bw Carcin-ogenicity Reported Irritation

C3H/HeJ (male) 80 2.5, 25 26, + 2.5%: No visible effects after wk11

21, 220, at 21 43% Recovery: substantially reversible
43a, 450, & 21% & 86.5: Scale, eschar formation, incrusted & keratinized nodules;
86.5 911 86.5% hyperkeratosis, scabbing, thickened subcutaneous tissue with pigmentation
NMRI (male) 80b 21.5, 25 217, – c
Effects observed at all dose levels with no or slight dose response: ulceration,
43.0, 444, scabbing, thickening with plication, increased inflammation, fibrosis,
85.0 919 hyperkeratosis, hyperplasia

a
Stop dose experiment: for 6 months followed by no exposure with termination at 131 weeks.
b
After 7 months 2-EHA treatment, half of each group were untreated for two months then treated with 12-O-tetradecanoylphorbol-l3-acetate (TPA) for 20 weeks,
and untreated up to study month 24.
c
2-EHA application for 24 months.

Table 4 V79 cells (according to OECD 476), which concluded 2-EHA did not
Spontaneous tumor profiles and immunology of mouse strains in 2-EHA dermal induce gene mutations at the HPRT locus. 2-EHA was also tested in an
studies. in vitro micronucleus test (MNT) in human lymphocyte cultures in
Strain Spontaneous tumor profile Immunology accordance with OECD Guideline 487, not showing an clastogenic or
aneugenic effect (Envigo, 2018). Thus, nBA and 2-EHA were not mu-
NMRI males: lung and lymphoreticular system tumors, H2q tagenic under the experimental conditions reported, adding to the
Harderian gland tumors, liver and adrenal gland
weight-of-evidence of non-genotoxicity for these materials.
neoplasms; females: lymphoreticular system, ovarian
and lung tumorsa
C3H/HeJ males: liver tumors; females: mammary tumorsb Tlr4Lps−d
4. Critical evalution of the 2-EHA carcinogenesis studies against
a
Rittinghausen et al. (1997); Bomhard and Mohr (1989); Bomhard (1993). contemporary criteria
b
Janvier Labs (2017).
4.1. Strain selection

were reported in the 39 animals evaluated at necropsy. In the
BaP + TPA group, 1 SCP, 24 SCC and 5 keratoacanthomas were con-
firmed. No skin tumors occurred after lifetime treatment with 2-EHA
(Mellert et al., 1994), in contrast to the findings in C3H/HeJ mice.
2-EHA did not act as an initiator in the study with NMRI mice.
Recent evaluation of the genotoxic and mutagenic properties of this
family of esters indicates these materials are not genotoxic in vivo (Suh
et al., 2018). Available point mutation tests have shown inconsistent
results with various acrylates. Most of those tests were performed prior
to OECD guidelines and appropriate data regarding cytotoxicity are not
given. Data from two current OECD guideline compliant experiments
conducted under GLP are provided as a supplement to this paper (Insert
Data in Brief). This includes (a) an in vitro mouse lymphoma (TK+/-)
assay (according to OECD 490), which concluded that under the ex-
perimental conditions described nBA did not induce forward mutations
or structural chromosome aberrations in L5178Y TK+/- cells in the
absence and the presence of metabolic activation; and (b) an in vitro
HPRT locus gene mutation assay utilizing cultures of Chinese hamster
Selection of the appropriate test species and strain is a critical initial
consideration in conduct of a rodent carcinogenicity study. Mice are a
suitable model based upon the previously mentioned aspects of rela-
tively short life span, their widespread use in pharmacological and
toxicological studies, and their susceptibility to tumor induction.
Neither the C3H/HeJ nor the NMRI strain has a high reported back-
ground incidence of dermal tumors which is the most basic criterion to
consider in strain selection (Table 4). However sufficient characteriza-
tion beyond tumor susceptibility is necessary and in the years since the
pre-guideline studies with 2-EHA were conducted, detailed character-
istics of the various mouse models are now more readily available, in-
cluding increased understanding of strain sensitivities, phenotypes, and
genotypes. The NMRI mouse is an outbred strain that readily ser-
oconverts, and carries the haplotype H2q (Janvier labs, 2017), asso-
ciated with susceptibility to collagen induced arthritis (Inglis et al.,
2007). It has a sex dependent profile of spontaneous tumors. In con-
trast, the C3H/HeJ, which was originally inbred for their high incidence
of mammary tumors, carries a mutation in the toll-like receptor 4

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Table 5
Criteria for chronic study dose level selection and identification of maximum tolerated dose (MTD).
Dose Level Criteriaa

Low Highest nonirritating dose

Intermediate Minimally irritating dose

High Macroscopic Microscopic

Achieves MTD Moderate erythema, scaling, mild edema, alopecia, thickening.
Exceeds MTD Ulcers, fissures, exudate/crust/eschar, nonviable tissues, anything leading to
destruction of the functional integrity of the epidermis (e.g., caking,
fissuring, open sores, eschar).
Epidermal hyperplasia, epidermal hyperkeratosis, epidermal parakeratosis,
adnexal atrophy/hyperplasia, fibrosis, minimal-mild spongiosis, minimal-mild
epidermal edema, minimal-moderate dermal edema, moderate inflammation
Interfollicular and follicular crust, mild to moderate microulcer, degeneration/
necrosis, moderate to marked epidermal edema, marked dermal edema, marked
inflammation

a
Criteria adapted from USEPA (1988); USEPA (1998).

(TLR4) gene making these mice less sensitive to endotoxin and highly
susceptible to infection by gram negative bacteria (Janvier Labs, 2017).
TLR4 deficiency also results in altered inflammatory cell infiltration,
differential cytokine production, and impaired wound closure (Chen,
2013). For substances like 2-EHA that cause chronic irritation-induced
wounding and inflammation, the underlying biological background of
the C3H/HeJ becomes an important consideration which will be dis-
cussed in more detail later in this paper.
4.2. Selection of dosing regimen
Proper dose selection is another critical guideline criteria in the
design of carcinogenicity studies. The consensus of experts at the EPA
workshop on design of dermal bioassay protocols agreed that regardless
whether the toxicity endpoint for estimating the MTD involves the skin
or is systemic, a 90-day dose finding study is necessary to determine the
MTD for a long term bioassay (USEPA, 1988). Currently, data from a
subchronic study (90 day repeated dose) with a sufficiently robust ex-
amination of clinical observations, measures of systemic toxicity, and
macroscopic and microscopic tissue examinations is recommended to
define the maximum tolerated dose (MTD) (OECDa, 2007; OECDb,
2007; USEPA, 1988, 1998). Specifically, the MTD should not shorten
the life of the animals, cause tissue necrosis or metabolic saturation
(Rhomberg et al., 2007). Supporting the need for macroscopic and
microscopic evaluation, McLaughlin et al. (1995) showed that data
from shorter duration studies or gross examination at termination alone
were insufficient to identify the MTD for irritation based on evaluation
of skin from subchronic dermal studies of acrylic acid in three mouse
strains. Detailed criteria for achieving or exceeding the MTD are in-
cluded in Table 5.
4.3. Assessment of the maximum tolerated dose
As mentioned earlier, dose selection for the initial screening study in
C3H/HeJ mice was based on a 2-week study and tissue examination
was limited to a gross evaluation (DePass et al., 1985). According to
contemporary criteria this is insufficient for proper MTD definition. A
subchronic dermal study was later conducted to assess the irritant
properties of 2-EHA solutions in two strains of mice, C3H/HeJ and
NMRI. Table 6A shows the results of the subchronic study performed as
a range finder for the chronic studies (and also the results of interim
evaluations of the application site during the conduct of the chronic
studies. In the C3H/HeJ chronic study, 12 weeks of treatment produced
eschar in 67 (84%), 49 (61%) and 52 (65%) of mice administered
86.5%, 43% and 21% 2-EHA, respectively (BASF, 1986), clearly ex-
ceeding the MTD and prompting the addition of a 2.5% group. Mac-
roscopic evidence of skin repair was noted after 11 weeks of exposure at
2.5%. Skin was essentially repaired even after 6 months exposure to
43% when left untreated for 18 months (Table 3). Regression of skin
irritation never occurred in the chronic dose groups at 21% and 86.5%
(Wenzel-Hartung et al., 1989) (Table 7).
To compare species sensitivity, the same dose levels: 21, 43, and
86.5% were used in the chronic study with NMRI mice. Macroscopic
and histopathological interim evaluation of the skin of 2 mice from
each dose group after 3 months showed scaling at the high dose, skin
lesions at the low and high dose, as well as erythema and skin thick-
ening with plication in all groups. Microscopic evidence of crust for-
mation and ulceration were also reported at 21%; lymphocyte in-
filtration, (BASF, 1992) hyperkeratosis, and hyperplasia were noted in
all groups (Table 6B). Based on these limited data, it appears that 21%
likely exceeds an MTD in the NMRI mouse. This persistent skin damage
is supported by information from the initiation promotion portion of
the study. Dosing with 2-EHA was stopped after 7 months and pro-
motion with TPA started after a 2 month treatment free period. At this
time 8 animals in the 21% group and 3 in the 86.5% group were re-
moved from the initiation promotion study due to persistent skin le-
sions (Mellert et al., 1994).
The MTD for dermal application was clearly exceeded at dose levels
> 21% in C3H/HeJ mice, and met or exceeded at this concentration in
NMRI mice. While exceeding an MTD limits the reliability of an

Table 6A
Interim evaluation of the application site following 3 months of 2-ethylhexyl acrylate treatment – percent incidence in range finding studies.
Mouse strain C3H/HeJ NMRI

Concentration % 0 86.5 0 21 86.5

a
Lesion type MTD Criteria number of animals = 10 10 5 5 5
Irritation-associated lesions Exceeds Scabbing – massive 0 10 0 0 0
(% affected) Achieves Scabbing – superficial 0 100 0 0 20
Thickened – subcutaneous 0 100 0 0 100
Pigmentation – subcutaneous 0 100 0 0 0
Epithelial hyperplasia 0 40 0 0 0

a
Maximum tolerated dose (MTD) criteria adapted from USEPA (1988); USEPA (1998).

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Table 6B
Interim evaluation of the application site following 3 months of 2-ethylhexyl acrylate treatment – percent incidence in chronic studies.
Mouse strain C3H/HeJ NMRI

a
Lesion type MTD Criteria Concentration % 0 2.5 21 43 86.5 0 21 43 86.5
number of animals = 80b 80b 80b 80b 80b 2c 2c 2c 2c
Irritation-associated lesions Exceeds Scaling nd 0 0 0 100
(% affected) Eschar 0 0 65 61 84 0 0 0 0
Ulceration nd 0 50 0 0
Crust formation 0 0 0 0 0 0 50 0 0
Achieves Epithelial hyperplasia nd 0 50 100 100
Thickened - subcutaneous 0 0 50 100
Epithelial hyperkeratosis 0 50 100 50

nd - not determined.
a
Maximum tolerated dose (MTD) criteria adapted from USEPA (1988); USEPA (1998).
b
Only macroscopic evaluation conducted.
c
Satellite group for histopathology.

Table 7
Selected skin lesions and tumors in male mice following lifetime 2-ethylhexyl acrylate treatment – Percent incidence.
Lesion type MTD Criteria1 C3H/HeJ Mice

2-Ethylhexyl acrylate (dose %) 0 2.5 21 86.5

number of animals = 80 80 80 80

Irritation-associated Exceeds Squamous (scaling) 0 0 31 38

(% affected) Eschar 1 9a 13 18
Squamous and eschar 0 0 21 20
Ulceration 0 0 4 1
Severe inflammation 0 0 0 1
Achieves Scabbing – superficial 1 14 51 29
Thickened - subcutaneous 0 99 70 85
Pigmentation - subcutaneous 0 53 68 90
Hyperkeratosis 1 9b 68 83
Epithelial hyperplasia 0 8 65 80
Tumor Squamous papilloma 0 0 6 13
(% affected) Squamous carcinoma 0 0 25 20
Melanosarcoma 0 0 9 11
Fibrosarcoma 0 0 6 0
Basal cell carcinoma 0 0 1 0
Cornu cutaneum (keratinous skin tumor) 0 0 1 4
Haemangioma 0 0 0 1c

Lesion type MTD Criteria1 NMRI Mice

2-Ethylhexyl acrylate (dose %) 0 21 43 86.5

number of animals = 41 40 39 38

Irritation-associated Exceeds Ulceration 5 18 26 31

(% affected) Scabbing 5 13 15 26
Achieves Lymphocyte infiltration 12 55 68 51
Increased macrophage 2 75 56 49
Dermal fibrosis 0 38 23 28
Hyperkeratosis 15 75 90 92
Epithelial hyperplasia 10 83 72 82
Tumor Squamous papilloma 0 0 0 0
(% affected) Squamous carcinoma 0 0 0 0
a
Focal or minimal.
b
Regressed after the 4-5th week of treatment.
c
Relevance to treatment is unclear.
1
Maximum tolerated dose (MTD) criteria adapted from EPA Health Effects Test Guideline OPPTS 870.4200 Carcinogenicity and USEPA workshop (1988).

identified hazard outcome for human health risk assessments, it cannot areata, (inflammation associated hair loss mediated by T-lymphocytes
fully explain the different tumor responses of 2-EHA in the C3H/HeJ (Gilhar et al., 1999), enhanced immune response (McElwee et al.,
versus NMRI mice. Despite the fact that the MTD was exceeded in both 2002), and cancer (Goldsworthy and Fransson-Steen, 2002; Yusuf et al.,
strains, tumors were only observed in C3H/HeJ mice. 2008).

4.4. Assessment of the biological background and unique sensitivity of the
C3H/HeJ strain
A factor that could contribute to the enhanced tumorigenic response
in the C3H/HeJ mouse is its mutated TLR-4 receptor, which imparts
susceptibility to development of autoimmune diseases like alopecia
4.4.1. Influence of TLR4 deficiency on the tumorigenesis process
Intraperitoneal injections of N,N-diethylnitrosamine to C3H/HeJ,
C57Bl6, and B6C3F1 mice showed the TLR4 deficient C3H strain de-
veloped more liver tumors and at an earlier onset (Goldsworthy and
Fransson-Steen, 2002). This was similarly demonstrated in skin with
dimethylbenzanthracene (DMBA) in C3H/HeJ mice vs. C3H/NeN

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S. Murphy et al.
Fig. 1. Proposed influence of strain difference on the mechanism of tumor formation after chronic dermal exposure to 2-ethylhexyl acrylate.
(TLR4 normal) mice; contact hypersensitivity appeared to play a pro-
tective role in the normal, but not the TLR4 deficient mice, suggesting
that IL and T cell types affect susceptibility to tumor formation (Yusuf
et al., 2008). The extent of dermal irritation by DMBA was not discussed
in this paper. Disturbances in the pro- and anti-inflammatory cytokine
balance results in an immune suppressive environment allowing tumors
to escape immune surveillance (Ouwehand, 2011). In humans, a non-
functional TLR4 allele has been associated with more frequent relapse
in breast cancer patients and increased risk for Heliobacter pylori gas-
tritis and cancer in Caucasian populations (Yusuf et al., 2009).
4.4.2. Immune response and wound healing effects on the tumorigenesis
process
Chen (2013) suggest that increased inflammatory cell content oc-
curs in wounds in the absence of functional TLR4; wounds heal more
slowly, and the number of inflammatory cells in the wound was slightly
increased, in keeping with the general notion that excessive cellular
inflammation can be detrimental to repair. The innate immune system
responds to multiple insults, including trauma and tissue necrosis, with
acute inflammation; if overwhelmed it can progress to the chronic state
of inflammation (Mittal et al., 2014). Pigmentation of the skin is as-
sociated with prolonged inflammation, which can stem from a variety
of sources, including allergic or contact dermatitis, laser therapy, or
burns (Patel, 2014). A majority of the skin samples from the C3H/HeJ
study reported pigmentation, implicating the occurrence of prolonged
inflammation caused by 2-EHA chronic treatment in this strain.
The association between chronic irritation and inflammation and
tumor formation has been recognized for over a century and inhibiting
inflammation can deter epithelial tumor formation (Marks et al., 2003;
Mueller, 2006). In the skin, the immune system responds to repair
tissue injuries/wounds. Neutrophils provide proteinases and reactive
oxygen species. They are also a source of the cytokines/chemokines
necessary for cell recruitment, activation and differentiation, which is
followed by cellular growth and proliferation. Macrophages provide the
growth factors and cytokines that control tissue repair. To complete the
healing process, remodeling of the extracellular matrix and angiogen-
esis are required (Mueller, 2006; Eming et al., 2014). Irritation or in-
flammatory reactions may alter cell kinetics or absorption, increase cell
turnover rate and increase the potential for faulty DNA replication, as
well as cause discomfort and increase stress (Rhomberg et al., 2007).
Successful tissue repair depends on orderly and timely repair which
involves the inflammatory response and multiple additional interac-
tions, including proliferation and migration of cells, matrix deposition
and tissue remodeling. Interruption or deregulation of any of the phases
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Toxicology Letters 294 (2018) 205–211
of wound healing leads to non-healing wounds (Eming et al., 2014).
Macrophages play an important role in the modulation of these
responses and the transition from acute inflammation to im-
munosuppression and healing (Koh and DiPietro, 2011). Early re-
sponses after injury are dominated by M1 macrophages, which produce
pro-inflammatory cytokines like IL1, IL-6, IL-12, and TNFα. Following
phagocytosis of apoptotic cells in the injured tissue, these macrophages
transform into a regulatory M2 phenotype, characterized by low IL-12
and high IL-10 expression (Filardy et al., 2010; Koh and DiPietro,
2011). This transition is crucial to resolve the acute inflammation re-
sponse and move toward repair (Landen et al., 2016). Altered gene
expression can disrupt the orderly interactions between inflammation,
angiogenesis, cell proliferation, apoptosis, etc. that allow normal cells
to be regenerated and damaged cells to be removed by the immune
system. With normal (acute) wound healing, inflammation lasts 1–3
days, proliferation 4–21 days and remodeling 21 days – 1 year (Landen
et al., 2016).
In adult skin the situation is complex and there appears to be a fine
line between “good” inflammation and “bad” inflammation as it per-
tains to repair. TLR4 may play a role in the innate immune system of
skin, which subtly harnesses inflammation so that wounds heal favor-
ably with resistance to infection and with appropriate restoration of
structure. Comparing TLR4 deficient and wild type mice, keratinocytes
at the wound edge of the former had decreased epidermal growth factor
(EGF) expression, which can affect their proliferation and migration.
Further, re-epithelialization occurred more slowly, and wounds con-
tained significantly more T cells on day 10, when these are declining in
wild type mice, suggesting increased inflammatory cells at discrete
times during skin wound healing (Chen, 2013).
In summary, chronic insult in excess of an MTD, together with
consequences of the extended timeline for macrophage transition, likely
exacerbates the tissue damage and creates an abnormally favorable
environment for tumorigenesis. This is illustrated in Fig. 1. Guidance on
evaluation of carcinogenicity studies recognizes the possibility that
mechanisms of toxicity active at high doses are not relevant to humans
exposed to lower doses, and the MTD should be based on an under-
standing of the mechanism of action to enhance the soundness of as-
sessments related to humans (OECD, 2002).
5. Conclusions
Using current guidance for setting the MTD, the 2.5% dose in the
C3H/HeJ mouse met the criteria for the MTD, and no skin tumors de-
veloped at this dose. The dose ranges in both the C3H/HeJ and NMRI
S. Murphy et al.
studies exceeded the MTD for skin integrity based on interim and
terminal evaluations of the application site skin condition. Nonetheless,
even studies with design flaws can provide useful information related to
hazard (Rhomberg et al., 2007), however tumor response information
in excess of an MTD should not be considered in a human hazard or risk
assessment paradigm. For the purposes of an appropriate hazard as-
sessment, 2-EHA did not cause or initiate dermal carcinogenesis in an
immune competent (NMRI) mouse model, and, even in an immune
compromised (C3H/HeJ) mouse model, did not induce skin tumors at
doses which did not exceed the MTD.
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References
Abel, E.L., Angel, J.M., Kiguchi, K., DiGiovanni, J., 2009. Multi-stage chemical carcino-
genesis in mouse skin: fundamentals and applications. Nat. Protoc. 4 (9), 1350–1362.
BAMM, 2014. Global Product Summary: 2-Ethylhexyl Acrylate. available online. http://
www.bamm.net/ethylhexyl-acrylate-eha/.
BASF, 1986. Dermal Oncogenicity Study of 2-Ethylhexyl Acrylate by Chronic
Epicutaneous Application in Male C3H Mice. Dec 9, 1986, Cited in the ECHA
Dissemination Dossier on 2-Ethylhexyl Acrylate.
BASF, 1992. Report on the Dermal Oncogenicity of 2-Ethylhexyl Acrylate in Male NMRI
Mice. Project Number 83C9/87032, Cited in the ECHA Dissemination Dossier on 2-
Ethylhexyl Acrylate.
Bomhard, E., 1993. Frequency of spontaneous tumours in NMRI mice in 21-month stu-
dies. Exp. Toxicol. Pathol. 45 (5-6), 269–289.
Bomhard, E., Mohr, U., 1989. Spontaneous tumors in NMRI mice from carcinogenicity
studies. Exp. Pathol. 36 (3), 129–145.
Chen, L., 2013. Toll-like receptor 4 has an essential role in early skin wound healing. J.
Invest. Dermatol. 133, 258–267.
DePass, L.R., Fowler, E.H., Meckley, D.R., Weil, C.S., 1984. Dermal oncogenicity bioas-
says of acrylic acid, ethyl acrylate, and butyl acrylate. J. Toxicol. Environ. Health 14
(2-3), 115–120.
DePass, L.R., Maronpot, R.R., Weil, C.S., 1985. Dermal oncogenicity bioassays of mono-
functional and multifunctional acrylates and acrylate-based oligomers. J. Toxicol.
Environ. Health 16 (1), 55–60.
Eming, S.A., Martin, P., Tomic-Canic, M., 2014. Wound repair and regeneration: me-
chanisms, signaling and translation. Sci. Transl. Med. 6 (265). http://dx.doi.org/10.
1126/scitranslmed.3009337. 265sr6.
Envigo, 2-Ethylhexylacrylate:Micronucleus Test In Human Lymphocytes In Vitro, un-
published report, 2018.
Filardy, A.A., Pires, D.R., Nunes, M.P., Takiya, C.M., Freire-de-Lima, C.G., Ribeiro-Gomes,
F.L., DosReis, G.A., 2010. Proinflammatory clearance of apoptotic neutrophils in-
duces an IL-12lowIL-10high regulatory phenotype in macrophages. J. Immunol. 185
(4), 2044–2050.
Gilhar, A., Shalaginov, R., Assy, B., Serafimovich, S., Kalish, R.S., 1999. Alopecia areata is
a t-lymphocyte mediated autoimmune disease: lesional human t-lymphocytes transfer
alopecia areata to human skin grafts on SCID mice. J. Invest. Dermatol. Symp. Proc. 4
(3), 207–210.
Goldsworthy, T.L., Fransson-Steen, R., 2002. Quantitation of the cancer process in
C57BL/6J, B6C3F1 and C3H/HeJ mice. Toxicol. Pathol. 30 (1), 97–105.
Gordon, S.C., Zimmerman, D.D., Griffith, F.D., 1991. Acute toxicity, genotoxicity, and
dermal carcinogenicity assessment of isooctyl acrylate. J. Toxicol. Environ. Health
34, 279–296.
Inglis, J.J., Criado, G., Medghalchi, M., Andrews, M., Sandison, A., Feldmann, M.,
Williams, R.O., 2007. Collagen-induced arthritis in C57BL/6 mice is associated with a
robust and sustained T-cell response to type II collagen. Arthritis. Res. Ther. 9 (5),
R113.
Janvier Labs, 2017. FICHE_RESEARCH_MODEL_NMRI.pdf. available online: https://
www.janvier-labs.com/tl_files/_media/images/FICHE_RESEARCH_MODEL_NMRI.
pdf.
Koh, T.J., DiPietro, L.A., 2011. Inflammation and wound healing: the role of the mac-
rophage. Expert Rev. Mol. Med. 13, e23. http://dx.doi.org/10.1017/
S1462399411001943.
211
Toxicology Letters 294 (2018) 205–211
Landen, N.X., Li, D., Stahle, M., 2016. Transition from inflammation to proliferation: a
critical step during wound healing. Cell Mol. Life Sci. 73 (20), 3861–3885.
Marks, F., Fuerstenberg, G., Neufang, G., Muelleker, K., 2003. Mouse skin as a model for
cancer chemoprevention by nonsteroidal anti-inflammatory drugs. Recent Results
Cancer Res. 163, 46–57.
McElwee, K.J., Hoffmann, R., Freyschmidt-Paul, P., Wenzel, E., Kissling, S., Sundberg,
J.P., Zöller, M., 2002. Resistance to alopecia areata in C3H/HeJ mice Is associated
with increased expression of regulatory cytokines and a failure to recruit CD4++
and CD8++ cells. J. Invest. Dermatol. 119 (6), 1426–1433.
McLaughlin, J.E., Parno, J., Garner, F.M., Clary, J.J., Thomas, W.C., Murphy, S.R., 1995.
Comparison of the maximum tolerated dose (MTD) dermal response in three strains
of mice following repeated exposure to acrylic acid. Food Chem. Toxicol. 33 (6),
507–613.
Megosh, L.C., Hu, J., George, K., O’Brien, T.G., 2002. Genetic control of polyamine-de-
pendent susceptibility to skin tumorigenesis. Genomics 79 (4), 505–512.
Mellert, W., Kühborth, B., Gembardt, C., Munk, R., 1994. 2-year carcinogenicity study in
the male NMRI mouse with 2-ethylhexyl acrylate by epicutaneous administration.
Food Chem. Toxicol. 32 (3), 233–237.
Mittal, M., Siddiqui, M.R., Tran, K., Reddy, S.P., Malik, A.B., 2014. Reactive oxygen
species in inflammation and tissue injury. Antioxid. Redox Signal. 20 (7), 1126–1167.
Mueller, M.M., 2006. Inflammation in epithelial skin tumours: old stories and new ideas.
Eur. J. Cancer 42 (6), 735–744.
OECD, 2002. OECD Environment, Health and Safety Publications. Series on Testing and
Assessment No. 35 and Series on Pesticides No. 14. Guidance Notes for Analysis and
Evaluation of Chronic Toxicity and Carcinogenicity Studies. 4 September 2002.
OECDa, 2007. Guideline for the Testing of Chemicals 452 Chronic Toxicity Studies. 7
September 2009.
OECDb, 2007. Guideline for the Testing of Chemicals 453 Combined Chronic
ToxicityCarcinogenicity Studies. 7 September 2009.
OECD, 2012. Guidance document 116 on the conduct and design of chronic toxicity and
carcinogenicity studies. Supporting Test Guidelines 451, 452 and 453, 2nd edition.
13 April 2012.
Ouwehand, K., 2011. The Skin Immune System: The Resourceful Army of Langerhans
Cells. PhD Thesis. Vrije Universiteit Amsterdam, pp. 144.
Patel, A.B., 2014. Postinflammatory hyperpigmentation: review of pathogenesis, pre-
vention, and treatment. Pigm. Int. 1, 59–69.
Proctor, D., Suh, M., Chappell, G., Borghoff, S., Thomason, C., Wiench, K., Finch, L., Ellis-
Hutchings, R., 2018. Development of an adverse outcome pathway (AOP) for for-
estomach tumors initiated by Non-genotoxic key events. Reg. Toxicol. Pharm. 96,
30–40. http://dx.doi.org/10.1016/j.yrtph.2018.04.016.
Rhomberg, L.K., Baetcke, J., Blancato, J., Bus, S., Cohen, R., Conolly, R., Dixit, J., Doe, K.,
Ekelman, P., Fenner-Crisp, P., Harvey, D., Hattis, A., Jacobs, D., Jacobson-Kram, T.,
Lewandowski, R., Liteplo, O., Pelkonen, J., Rice, D., Somers, A., Turturro, W., West,
S., Olin, S., 2007. Issues in the design and interpretation of chronic toxicity and
carcinogenicity studies in rodents: approaches to dose selection. Crit. Rev. Toxicol.
37, 729–837.
Rittinghausen, S., Kaspareit, J., Mohr, U., 1997. Incidence and spectrum of spontaneous
neoplasms in Han: NMRI mice of both sexes. Exp. Toxicol. Pathol. 49 (5), 347–3499.
Singh, M., Suman, S., Shukla, Y., 2014. New enlightenment of skin cancer chemopre-
vention through Phytochemicals: In Vitro and In Vivo Studies and the Underlying
Mechanisms. Biomed. Res. Int. 18 243452.
Suh, M., Proctor, D., Chappell, G., Rager, J., Thompson, C., Borghoff, S., Finch, L., Ellis-
Hutchings, R., Wiench, K., 2018. A review of the genotoxic, mutagenic, and carci-
nogenic potentials of several lower acrylates. Toxicology. http://dx.doi.org/10.1016/
j.tox.2018.04.006. Available online 22 April 2018.
Thompson, C., Suh, M., Chappell, G., Borghoff, S., Ellis-Hutchings, R., Wiench, K., Finch,
L., Proctor, D., 2018. Assessment of the Mode of action underlying development of
forestomach tumors in rodents following Oral exposure to ethyl acrylate and re-
levance to humans. Regul. Toxicol. Pharmacol. 402-403, 50–67. http://dx.doi.org/
10.1016/j.yrtph.2018.05.006.
USEPA, 1988. Summary of the Second EPA Workshop on Carcinogenesis Bioassay via the
Dermal Route, May 18-19, 1988 Research Triangle Park NC. EPA560/6-89-003.
USEPA, 1998. Health Effects Test Guidelines Carcinogenicity OPPTS 870.4200. EPA
712–C–98–211.
Wenzel-Hartung, R.F., Klimisch, H.J., Brune, H., 1989. Dermal oncogenicity study of 2-
ethylhexyl acrylate by epicutaneous application in male C3H/HeJ mice. J. Cancer
Res. Clin. Oncol. 115 (6), 543–549.
Yusuf, N., Nasti, T.H., Long, J.A., Nassemuddin, M., Lucas, A.P., Xu, H., Elmets, C.A.,
2008. Protective role of Toll-like receptor 4 during the initiation stage of cutaneous
chemical carcinogenesis. Cancer Res. 68 (2), 615–622.
Yusuf, N., Nasti, T.H., Meleth, S., Elmets, C.A., 2009. Resveratrol enhances cell-mediated
immune response to DMBA through TLR4 and prevents DMBA induced cutaneous
carcinogenesis. Mol. Carcinog. 48 (8), 713–723.