Cutaneous and Ocular Toxicology
ISSN: 1556-9527 (Print) 1556-9535 (Online) Journal homepage: http://www.tandfonline.com/loi/icot20
The importance of minipigs in dermal safety
assessment: an overview
Alain Stricker-Krongrad, Catherine R. Shoemake, Jason Liu, Derek
Brocksmith & Guy Bouchard
To cite this article: Alain Stricker-Krongrad, Catherine R. Shoemake, Jason Liu, Derek
Brocksmith & Guy Bouchard (2016): The importance of minipigs in dermal safety assessment:
an overview, Cutaneous and Ocular Toxicology, DOI: 10.1080/15569527.2016.1178277
To link to this article: http://dx.doi.org/10.1080/15569527.2016.1178277
Published online: 10 May 2016.
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ISSN: 1556-9527 (print), 1556-9535 (electronic)
Cutan Ocul Toxicol, Early Online: 1–9
! 2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/15569527.2016.1178277
REVIEW ARTICLE
The importance of minipigs in dermal safety assessment: an overview
Alain Stricker-Krongrad1, Catherine R. Shoemake1, Jason Liu1, Derek Brocksmith2, and Guy Bouchard1
Sinclair Research Center, Columbia, MO, USA and 2Sinclair BioResources, Columbia, MO, USA
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1
Abstract
Keywords
The use of miniature swine as a non-rodent species in safety assessment has continued to
expand for over a decade and their use has become routine, particularly in pharmacology as a
model for human integumentary diseases. Translational preclinical swine study data are now
favorably compared and contrasted to human data, and miniature swine models provide
important information in dermal safety assessment and skin pharmacology. For example, the
miniature swine model has been well-accepted for cutaneous absorption and toxicity studies
due to swine integument being morphologically and functionally similar to human skin.
Subsequently, this model is important to dermal drug development programs, and it is the
animal model of choice for assessment of dermal absorption, local tolerance and systemic
toxicity following dermal exposures. In conclusion, the miniature swine model has an important
role to play in the safety assessment of pharmaceutical products and in multiple aspects of
human dermal drug development.
Dermal drug safety, dermal pharmacology,
dermal toxicology
Introduction
The study of pigs as medical models is recorded from as
early as the time of Galen in 2nd century A.D.1 and their
study as biomedical models has slowly progressed to modern
times. During the past half century, pigs have been used in
preclinical dermal toxicology, dermal pharmacokinetics
(PK), dermal phototoxicity, dermal wound healing studies
and a broad array of other biomedical research applications2,3. Now in modern times, particularly over the past 30
years, the use of swine as biomedical models has grown
exponentially.
Swine have been used extensively in dermal research
because of the comparability of their integument to that of
humans (cf. Figure 1). Reviews of the use of swine in such
studies have been previously published3–7. In the field of
toxicology, swine skin has been used for acute and repeat dose
dermal toxicology, dermal absorption, allergic contact dermatitis, phototoxicity and photosensitization studies. Models
have been created both in vivo as well as in vitro with skin
membranes and grafts. Both miniature and domestic breeds
have been used for these types of studies; however, miniature
breeds such as Sinclair, Gottingen, Yucatan and Hanford may
be more advantageous because of their smaller size at sexual
maturity. Table 1 presents some primary aspects to consider
when selecting miniature swine versus domestic swine. Using
these miniature breeds allows investigators to conduct
Address for correspondence: Alain Stricker-Krongrad, PhD, Sinclair
Research Center, 562 State Road DD, Auxvasse, MO 65231, USA.
E-mail: [email protected]
History
Received 4 March 2016
Revised 29 March 2016
Accepted 11 April 2016
Published online 4 May 2016
experiments in mature (rather than pediatric) animals with a
consistent size and health status as compared to farm pigs.
Each breed may be utilized in some aspect of dermal
toxicology.
Selection of animal species for toxicology
Selection of the most appropriate animal species for preclinical toxicology testing during human drug development can
be a challenge; seeking advice from regulatory bodies such as
the U.S. Food and Drug Administration (FDA), the European
Medicines Agency (EMA), and the Organization for
Economic Cooperation and Development (OECD) is encouraged. Similarity to man with regard to anatomy and physiology is certainly important, but highly specific translational
predictive value for safety of the drug class under consideration is optimal. Miniswine have increased in rank as an
animal model in recent years; in some areas, the swine model
is preferred over non-human primates (NHP) or dog models to
translate directly into man8–10. Not all miniswine translational
research is yet fully realized or openly published as some data
remain confidential and are sequestered in private archives.
Under most conditions, swine data are evaluated in conjunction with data from other species for making drug development and regulatory decisions.
Selection of the most desirable species for dermal
toxicology additionally has many factors requiring consideration, including skin surface area, animal housing and
handling, per diem expenses, and predictability. For example,
miniswine, and pigs in general, have limited neck and
leg mobility and are unable to lick or scratch their backs.
2
A. Stricker-Krongrad et al.
1
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3
Cutan Ocul Toxicol, Early Online: 1–9
2
4
5
6
7
8
Figure 1. Comparative histological (H&E staining) examination of integument of seven species of mammals including two lineages of minipigs. 1:
human, 2; 2: cynomolgus monkey, 10; 3: Yucatan minipig, 10; 4: Hanford minipig, 2; 5: Beagle dog, 10; 6: rabbit, 2; 7: guinea pig, 20; 8:
mouse, 20. Hair follicle (H), Primary hair (PH), Secondary hair (SH), Sebaceous gland (S), Apocrine gland (A), Eccrine gland (E).
They can, however, scratch dorsal dermal application areas
(DAA) on surrounding structures, therefore test sites are
generally protected with covers or wraps, though some studies
do call for the sites to be left intentionally uncovered.
Beneficially, miniswine are easily trained and socialized, and
are able to be placed into slings for short periods of time, not
to exceed 3 h, without overt stress. Certain processes and
factors also must be closely monitored in order to avoid
Minipig in dermal safety assessment
DOI: 10.1080/15569527.2016.1178277
3
Table 1. Miniature versus domestic swine for toxicology.
Porcine
model
Size/sexual
maturity
Growth during
studies
Ease of
handling
Miniswine
Smaller, reaches
sexual maturity earlier
Slow, requires less
candidate drug
Good
Domestic
swine
Larger, reaches
sexual maturity later
Fast, requires more
candidate drug
Poor, unless
very young
Table 2. Predictive value of swine studies for human drug safety and
efficacy.
Controlled genotype
Closed herds or
inbred, lineages
outbred to greatest extent, wellcharacterized
Inbred or crossbred
breeds
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Good
Acceptable
Poor
Safety
Efficacy
17 (63%)
7 (26%)
3 (11%)
7 (100%)
0 (0%)
0 (0%)
topical test article cross contamination between treatment
groups, including blood collection, biopsies, pen changing,
caging sanitization procedures, dosing procedures, gloves,
slings, worker clothing and laundry.
Predictability of miniswine data for preclinical safety
Ganderup et al.11 reviewed miniswine safety and efficacy data
on 43 marketed drugs with previously reported adverse
responses. Fifty-eight percent of the reviewed drugs had a
dermal indication, and 27 drugs had both human and
miniswine data to enable a comparison. Overall, the predictive value (PV) of miniswine safety and efficacy studies to
human outcomes were 89% and 100%, respectively (Table 2).
The results of this review support the value of the miniswine
model for preclinical safety.
Comparative dermal anatomy and physiology
As with all animal models there are both similarities and
differences between swine and humans. Aside from a few
minor differences, the skin of swine is known to be very
similar to human skin5,12,13. Macroscopically, the swine is a
relatively hairless animal with a fixed skin that is tightly
attached to the subcutaneous tissues. Swine skin surface
character, thickness, layers, pigmentation, turnover kinetics,
number of hairs and hair follicles, blood flow, and variations
by gender, age and body region are generally representative of
humans. The skin pH of the pig is slightly higher than human
skin, having a less acidic mantle, and the hypodermal adipose
can be thicker in older overfed swine. In juvenile swine, hairs
and follicles can occasionally be found in triads; the overall
percentage of triad formations is low and they tend to outgrow
this pattern. Hair follicles contain an intrafollicular muscle
which contributes to the erection and rotation of hair shafts in
addition to the arrector pili muscles13,14. Seasonal shedding is
not generally an issue for miniswine when they are housed
indoors under controlled photoperiod and temperature.
Limitations
Yes or no depending on breed/
lineage
Subchronic and chronic
studies possible
Yes or no depending on source
Studies beyond 28 days inlive or 3 months age is
not recommended due to
size (expect 4100 kg at 4
months of age)
Table 3. Histological layers of swine skin and underpinnings.
Epidermis
Predictive value
Microbiologically
defined
Stratum corneum
Stratum lucidum
Stratum granulosum
Stratum spinosum
Stratum basale
Basement membrane
Dermis
Underpinnings
Papillary layer
Reticular layer
Hypodermis (adipose)
Fascia and muscle
Miniature breeds can either have pigmented or non-pigmented
skin, and the Sinclair and Yucatan breeds may be procured in
a spotted variety in which both types of skin are available on
the same animal4,15,16. Additionally, miniswine and humans
have similar body surface areas, making miniswine more
comparable to humans than smaller animals such as rodents17.
While humans, NHPs and swine all have fixed skin that is
adherent to underlying structures, rodents – rats, mice and
guinea pigs – have loose, poorly adherent skin18. This
‘‘tightness’’ of skin is related to the subcutaneous connective
tissue structures and the epidermal–dermal connection.
Porcine and human skin is similar in appearance on magnified
H&E-stained images. Normal pig skin has been described
both microscopically and ultrastructurally12,14. The histologically evident layers of miniswine skin and underpinnings are
presented in Table 3. The cellular epidermis is similar in
thickness, melanin distribution appears the same, and the
epidermis undulates with many rete pegs projecting inward
and correspondent dermal ridges projecting upward. The
collagen of both swine and human dermis appears glassy and
eosinophilic and has a compact criss-crossed collagen meshwork. In contrast, the loose-skinned species have much
thinner epidermis and fewer rete pegs. The dermis of looseskinned species is also less eosinophilic and the collagen
appears less dense.
Hair follicles and sebaceous glands have been recognized
as important pathways for percutaneous penetration of
topically applied lipophilic drugs via the pilosebaceous
route. Increased hair follicle density (Tables 4 and 5), as is
the case with most animal models outside of humans and
swine, can to a degree influence skin drug absorption for
selected compounds with unique chemistry19. In a comparison across species, it was found that NHP skin tends to have
relatively many hair follicles while the mouse appears to have
the most hair follicles. Hairless does not mean there are no
follicles, though; the hairless rat has a follicle density of
4
A. Stricker-Krongrad et al.
Cutan Ocul Toxicol, Early Online: 1–9
Table 4. Comparative pelage: hair follicle density.
Species
Area of skin
Human
Pig
Rat
Mouse
Hairless mouse
Abdomen
Back
Back
Back
Back
# hair follicles/cm2
11 ± 1
11 ± 1
289 ± 21
658 ± 38
75 ± 6
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Table 5. Human versus swine epidermal turnover, pH and skin hair
density.
Parameter
Human
Swine
Epidermal turnover (days)
Epidermal pH
Hair density (per cm2)
27–28
5
11
30
6–7
11
75 per cm2 and the nude guinea pig has many large empty hair
follicles evident as well. Humans and miniswine have less
hair than most animals, approximately 11 follicles per cm2,
therefore follicular uptake is less significant. In miniswine,
the range of hair counts or follicle density per unit area does
not vary much across lineages or from human counts, thus it
is not a major contributor to significant differences in
percutaneous absorption. Additionally, the interfollicular
area predominates over the follicular area by several-fold
for both swine and humans. The human follicular area is
typically only 0.1% of the total skin surface area, but the
follicular area on the face and scalp can be 10% of the total
face and scalp surface area. Finally, enhanced follicular
uptake of highly lipid soluble drugs is a unique prospect
applicable to only a few drug classes. Swine hair follicles are
typically larger than those of humans (177 mm versus 70 mm)
but this has minimal, if any, effect on uptake. Swine follicles
are reported to be no more penetrable than the epidermis in
general20.
Sebaceous glands as well as apocrine and eccrine sweat
glands of swine have differences in function, number and
location from those of humans. In pigs, apocrine sweat glands
are extensive but do not significantly contribute to sweating or
thermoregulatory functions, and eccrine sweat glands are
limited to the snout and carpal glands. There is also a mental
gland on the ventral chin which is a mass of apocrine and
sebaceous glands with tactile hairs. The secretions onto the
skin may contribute to preventing fluid loss but do not have a
sweating function like that of humans. Consequently, pigs
thermoregulate by blood flow modulation and by either
finding shade or wallowing in mud5,6,14.
Correlated skin drug penetration (going into skin) or
permeability (transdermal systemic absorption) are important
considerations when selecting a dermal animal model5,21–23.
Each skin layer plays a role in transdermal penetration of test
substances and must be considered during product development. The pH of the skin is 6–7 in swine and approximately 5
in humans. The cellular turnover rate in swine skin is
approximately 28–30 days, which is similar to humans5,14.
The skin of the pig is thicker and somewhat less vascular
overall than human skin. The skin thickness is especially
pronounced on the dorsal surface of the neck and back of
sexually mature animals and particularly in some breeds such
as the Yucatan. The thinnest skin is located on the ventral
abdomen and pinnae. There are approximately 60–75 capillary loops/mm2 in human and pig skin and approximately
0.7 m of blood vessel length/cm2 19. Blood flow varies by
anatomical region and is highest in the ventral abdomen at
approximately 18 ml/min/100 g and lowest in the dorsum and
buttocks at approximately 3 ml/min/100 g. The capillaries are
more involved with body temperature regulation than in
humans but otherwise they perform the same essential
functions of nutrient transport, waste products removal and
blood pressure regulation6,13,14. The cutaneous blood supply
and sequence of events in wound healing are similar to that in
humans24.
Epidermal and dermal measurements have been made in
both domestic and miniature breeds and were found to be
comparable between animals of a similar age4,6,23. Humans
have been described as having an epidermis of 70 mm (50–
120 mm) in thickness with a stratum corneum (SC) of 0.01 mm
and a dermis of 2.28 mm2 5. Swine epidermis is 70–140 mm
thick and composed of the following layers from outside to
inside: SC, stratum lucidum, statum granulosum, stratum
spinosum and stratum basale. The epidermis ends at the
epidermal–dermal junction12,13. In one study, sexually mature
4–6 mo Yucatans had a full-thickness measurement ranging
from 1.5 mm on the flank to 2 mm on the dorsum4. The
epidermis ranged from 0.04 to 0.1 mm, also depending upon
the location, and the SC was 0.02 mm with 15 layers. Juvenile
pigs (1.5–3 mo) were reported to have an epidermis of 0.05–
0.065 mm, a SC of 0.01 mm and a dermis of 1.15–1.56 mm
thick3,4,6,23.
In swine, the SC contains approximately 15 layers of
keratinized stratified squamous epithelium which is continuously desquamated. Beneath it is the thin translucent stratum
lucidum with keratinized cells devoid of nuclei. This layer
contains protein-bound phospholipids and eleidin, which is
especially abundant in thick, hairless skin. The stratum
granulosum is the next layer and consists of cells containing
keratohyalin and lamellar granules that release lipid by
exocytosis into the intercellular space. Hydrolytic enzymes
interact with the lipids resulting in an intercellular lipid
matrix. This lipid matrix between keratinized cells is both the
primary barrier against and the pathway for penetration of
topical drugs. The physical properties of this matrix contribute to the similarities of transdermal penetrance in swine to
humans. The stratum spinosum is immediately below this
layer, then the stratum basale which consists of columnar or
cuboidal cells that have the dual function of attaching to the
dermis and producing new epidermal cells. There are
approximately four viable cell layers in the stratum basale
of swine. Non-keratinocytes present in the epidermis are
melanocytes, Merkel cells and Langerhans cells5,13,14.
The epidermal–dermal junction provides the basement
membrane to the epidermal cells and the connection to the
dermis. There are eight separate antigenic epitopes in this
structure. Six of these cross react with those of humans:
laminin, type IV collagen, fibronectin, GB3, BP and EBA.
The remaining two, L3d and 19-DEJ-1 do not. This structure
is ultrastructurally similar to humans and invaginates into the
dermis with epidermal pegs and dermal papilla. In addition to
Minipig in dermal safety assessment
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DOI: 10.1080/15569527.2016.1178277
its adherence and maintenance functions, this structure is also
a selective barrier for molecular restriction and transport,
wound healing, and immunological function5,13,14.
The dermis, or corium, is composed of dense irregular
connective tissue containing elastic, collagen and reticular
fibers in amorphous ground substance. This layer contains
numerous cellular and adnexal structures. Commonly found
cells are fibroblasts, mast cells, plasma cells, macrophages,
chromatophores and fat cells. Blood vessels, lymphatics and
nerves are predominantly located in this layer, as well as
sweat glands, sebaceous glands, and hair follicles with their
associated intrafollicular and arrector pili muscles5,13,14.
The hypodermis, or subcutis, is a layer of loose connective
tissue which produces fascia and elastic fibers connecting the
skin to muscle. The subcutaneous fat, or panniculus adiposus,
is located in this region. In boars, this layer becomes traversed
with collagen fibers which provide a protective shield on the
dorsolateral aspects of the body5,13,14.
In summary, the dermal anatomic and physiologic
similarities between pigs and humans include a sparse hair
coat, a relatively thick epidermis, epidermal turnover kinetics,
lipid composition, lipid biophysical properties, and arrangement of dermal collagen and elastic fibers. The differences
are the interfollicular muscle, the distribution and function of
apocrine versus eccrine sweat glands, thickness of the SC, the
basement membrane epitopes, and Cytochrome P-450 biotransformation isoenzymes3,5,6,24. Also, miniature swine offer
advantages over domestic pigs for preclinical applications,
considerations of which are presented in Table 12.
5
follicles, apocrine sweat glands, and the subcutaneous layer of
adipose, blood vessels, nerves, and the beginnings of skeletal
muscle.
Background histopathology
As shown in Table 6, inflammation and mononuclear
infiltrates were the most common incidental background
findings across the four most common miniswine breeds in
the US: Göttingen, Yucatan, Hanford and Sinclair. Yucatan
miniswine exhibited mononuclear infiltrates in 5.6% of the
animals. The Hanford strain exhibited a variety of types of
inflammation, ranging from acute or chronic dermatitis in
13.5% of the animals to chronic perifollicular inflammation
and multifocal lymphohistiocytic inflammation, each in just
under 2% of the tested population. The Göttingen breed had a
broader range of findings, including crusts (9.1%), hyperkeratosis and parakeratosis (4.9%), and epidermal and
subepidermal edema (6.3%). They also had mononuclear
and inflammatory cells present in 13.3% of the population.
Dermal toxicology models
The landscape of dermal study models and applications
is expanding, and Hanford, Sinclair, and Yucatan miniswine
are each utilized in some aspect of dermal toxicology
testing or dermal research2,3,6,24. Below are selected model
descriptions.
Dermal toxicology
Distribution of skin thickness
Full-thickness skin is composed of the SC, cellular epidermis
and dermis. It is well recognized that skin thickness varies
from region to region of the body and the density of hair
follicles varies greatly by region as well18. The Yucatan
minipig is no exception, as demonstrated in a study evaluating
body surface sites for 18 animals including neck, back, flank
and abdomen. The dermis made up the majority of the
thickness of Yucatan skin (92–99%). The Yucatan epidermis
(stratum corneum plus cellular epidermis) made up 1.0–8.0%
of full-thickness skin. The full-thickness epidermis ranged
from 62.36 mm on a 10.3-month-old castrated male flank to
134.15 mm on the neck of this same castrated male. Group
mean full-thickness skin ranged from 955.83 mm (0.95 mm)
on the back of 3.5-week-old females to 5666.32 mm (5.6 mm)
on the neck of a 10.3-month-old castrated male. Cellular
epidermis thickness ranged from 32.80 mm on the abdomen to
140.93 mm on the flank. SC thickness ranged from 6.23 mm on
the abdomen to 88.13 mm on the neck. Dermis thickness
ranged from 587.29 mm on the abdomen to 6741.67 mm on
the neck.
The thickness of the SC and epidermis plays a significant
role in affecting absorption of percutaneously applied drugs as
the near surface vascular supply underlies the epidermis
between the epidermal rete pegs in the uppermost dermis. The
thickness of the SC influences the resistance of the skin to
physical and chemical trauma and potentially to transdermal
drug delivery. The thickness of the dermis may also have an
impact on dermal absorption as the dermis depths contain hair
In light of the morphological and physiological similarities
between human and porcine skin that exceeds other laboratory
animal species, the miniature pig is a preferred model for
evaluating the safety profile of dermally applied xenobiotics.
Swine as a model in toxicity testing of pharmaceuticals and
other chemicals is now being well-accepted by Japan, EU,
Canada and USA regulatory agencies. Miniswine are also an
accepted second species for GLP toxicology/safety assessment25; the OECD 409 Guideline even lists swine and
minipigs as options for the second non-rodent species in
toxicology testing. In most cases, swine should be selected as
a primary species over dogs and rabbits as a dermal
toxicology model26. Young adult, 3–6-month-old Hanford
miniswine are most commonly used. Dermal studies in
miniswine allow the evaluation of both local and systemic
toxicity, and miniswine normal reference data is readily
available.
Transdermal absorption
In general, the pig is accepted as an appropriate model for
topical agent testing and skin penetrance is second only to
macaques in its similarity to humans for both lipophilic and
hydrophilic drugs.
Human permeability is higher than pigs for most compounds tested27, but miniature swine are still a recognized
predictive model for human drug candidate dermato-pharmacology studies28.
Penetrance of the skin by topical agents may be due to
intercellular, pore, interfollicular and/or skin breaks pathways.
6
A. Stricker-Krongrad et al.
Cutan Ocul Toxicol, Early Online: 1–9
Table 6. Most common incidental dermal histopathologic findings in control minipigsa.
Breed
Findings
Male
Female
Crust, focal, minimal to slight
Hyper/parakeratosis, focal to diffuse, minimal
Epidermal/subepidermal edema, focal, minimal to slight
Mononuclear/inflammatory cells, focal, minimal to moderate
n ¼ 143
4.2%
2.1%
1.4%
6.3%
n ¼ 143
4.9%
2.8%
4.9%
7.0%
Mononuclear infiltrates, focal, minimal
n ¼ 18
5.6%
n ¼ 21
0.0%
Acute inflammation, dermis, minimal
Chronic inflammation, dermis, minimal
Chronic inflammation, perifollicular, minimal
Lymphohistiocytic inflammation, multifocal, mild
n ¼ 60
0.0%
6.7%
0.0%
0.0%
n ¼ 59
1.7%
5.1%
1.7%
1.7%
Göttingen
Yucatan
Hanford
a
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No dermal histopathologic findings observed in the Sinclair minipig.
The lipid matrix within the stratum granulosum also affects
penetrability. Absorption may be variable depending upon
temperature, humidity, the skin condition, the surface area of
the application, the location on the skin and whether the area
is covered or uncovered. The properties of the agent and its
vehicle are also important. Sometimes transdermal drug
delivery also includes the technique of iontophoresis, which
involves the use of electrical current to enhance penetration of
drugs that ordinarily would not be permeable. With this
technique, charged drugs are transported after applying an
opposing electrical field. The pig ear has been used as a
predictive model because it is relatively thin and highly
vascularized, but transdermal absorption has been performed
on the ventral abdomen and dorsum as well. In the caudal
ventral abdomen, there is opportunity to study absorption in
areas of direct cutaneous blood supply in the region of the last
nipples versus the musculocutaneous blood supply cranial to
that region23,29,30.
Body surface area
Dermal maximum tolerated dose (MTD or dose escalation)
studies are common in miniswine22. Their dermal surface area
is adequate for these studies, and changes during growth have
been investigated to determine the potential effect upon the
dose per unit surface area and dose per unit body weight.
Several methods for calculating body surface area of domestic
swine have been published, including those by Spector31 and
Wachtel et al.32. The Spector method is highly respected, thus
it was selected and applied to miniswine in a study where the
total body surface areas (TBSA) of 32 growing male and
female Hanford miniature swine were calculated using body
weight at 0, 8, 12 and 18 weeks of age. Changes in TBSA
were compared to a DAA which was 5 cm 5 cm at the
beginning of the study. Changes in DAA, TBSA and body
mass are relevant to correlating doses topically applied and
systemically delivered in transdermal drug uptake studies. If
the DAA fails to grow proportionally to the body surface area
or to body mass, then the dose delivered on a per kilogram or
per surface area basis may vary across time. Wachtel et al.32
suggested that the formula for TBSA derived for domestic
swine is not applicable to miniswine. The Wachtel equation
SA (M2) ¼ 0.121 W(.575) was suggested to be a more
accurate, quick assessment of TBSA of miniswine. Therefore,
the Wachtel method, as well as Brody’s equation
(SA cm2¼970 W.633), was compared to the Spector
Method results.
Using the Spector formula for TBSA, the mean (±standard
deviation) DAA to TBSA ratio (DAA:TBSA) for week 0 and 8
for a control application area was 0.46%±0.04 and
0.51%±0.06, respectively. After week 8, subsequent periodic
measurements of the DAA:TBSA in 20–35 kg miniswine
remained steady or essentially unchanged, suggesting proportional changes in growth of both DAA and TBSA.
The Brody and Wachtel methods resulted in slightly
different absolute TBSA and DAA:TBSA values but in the
end all three methods were moving proportionally over the
four measurement time periods. Comparable TBSA and
DAA:TBSA data suggested the Spector Method was a valid
choice. The correlation of the three TBSA calculation
methods was 40.99; all three methods were comparable
with 510% difference. No actual TBSA reference value or
skin area measurements were available to ascertain accuracy
and precision of the formulas.
Skin stripping technique for dermal penetration
Tape stripping is a simple and effective method for removing
the SC and is commonly employed during in vivo studies
investigating the percutaneous penetration and disposition of
topically applied candidate drugs. Skin can be prepared by
washing it with gentle detergent; it can also be prepared by
adhesive tape stripping it to reduce the thickness of the SC for
dermal penetration studies. One study was performed with the
objective to assess the remaining thickness of the SC
following 0, 10, 20, 30, 40 and 50 repetitions of tape
stripping of skin on three young adult, male Yucatan
miniature swine weighing 33–36 kg. Following clipping of
the pelage over the dorsal lumbar and thoracic areas, six 5 cm
by 5 cm sites were demarcated and skin was stripped using
1.8 mm clear acrylic adhesive tape applied with uniform, firm
pressure. The results of analysis by light microscopy showed
an inverse pattern of SC thickness to the number of tape
stripping repetitions. After 20 strippings, the number of
layers was reduced from 11–15 down to 2–6 and 50 passes
were required to remove nearly all SC. No immediately
Minipig in dermal safety assessment
DOI: 10.1080/15569527.2016.1178277
detectable underlying changes of the epidermis or dermis
were observed. These data demonstrate that skin can be
stripped of SC in a linear fashion based upon repetition of the
technique.
Clinical evaluation of topical reactions
Draize scoring, developed by Draize33, provides a method of
assessing the degree of inflammation based on quantifying
the values that are at risk of interpretation bias or being
considered insignificant. Originally developed for use in
rabbits, it has been modified for use in both swine and
humans, thus is often referred to as the Modified Draize
Score. In dermal studies, the values that need quantified
analysis are erythema and edema. Erythema and edema are
each graded on a scale ranging from 0 to 4. For edema this
7
ranges from ‘‘no edema’’ at a score of 0 to ‘‘severe edema
raised 41 mm and extending beyond the area of exposure’’ at
a grade of 4 and for erythema this ranges from ‘‘no
erythema’’ to ‘‘severe erythema or slight eschar formation’’.
The grades 1, 2 and 3 for each value are slight, well-defined
and moderate or severe, respectively.
Type of vehicles for dermal topical applications
Based on information drawn from studies conducted over the
past 15 years, there is a wide variety of product combinations
that may serve as vehicles for pharmacology testing. Of the 11
studies selected for this report, nine were performed on
Hanford miniswine, two were performed on Sinclair miniswine and no specific vehicle was used more than once. As
shown in Table 7, vehicles that resulted in no adverse
Table 7. Dermal vehicles and their tolerability when applied to miniswinea.
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Vehicle
Duration
(days)
Dehydrated ethanol 200 Proof;
Hexylene glycol; Dimethiconol
blend 20; Hydroxypropylcellulose;
Anhydrous citric acid
Purified water; NaCl, KCl, L-arginine
HCl, NaOH; Emollients, emulsifiers and thickening agents
28
Methyl- and Propylparaben
50:50 Ethanol:Propylene glycol; BHA
and BHT
91
112
Gelatin phosphate buffer
Purified water; Denatured alcohol
190-proof; Propylene glycol;
NaOH solution; Phenoxyethanol;
Emollients, emulsifiers, and
thickening agents
PEG 400
Soybean, coconut, and mineral oil;
Beeswax; Stearic acid; Emollients,
emulsifiers, and thickening agents
Purified water; Olive oil; Shea butter;
Emollients, emulsifiers, and
thickening agents; Methyl- and
propylparaben
Ethanol 190 proof; Deionized water;
Glycerin; Propylene glycol;
Salicylic acid; EDTA; Emollients,
emulsifiers, and thickening agents
Water; Propylene glycol; Ethanol 200
proof; NaOH solution; Emollients,
emulsifiers, and thickening agents;
Phenoxyethanol
7
28
210
90
21
14
92
91
Tolerability
Percent
affected
Pathology
Number of
animals
Breed
10
Sinclair
Well-tolerated
20%
Normal
Severe persistent dose-site
erythema beginning on
day 2
33%
6
Sinclair
Well-tolerated
Very mild erythema on first
day, resolved
0%
67%
12
12
Hanford
Hanford
Slight erythema, resolved
10%
Three with chronic-active
superficial dermis
inflammation, one with
chronic subcutis inflammation as well; two had
bacterial infection, grade
1–3 hyperkeratosis and
surface exudate
Normal
Grade 2 neutrophilic
inflammation on one
male, Grade 2 mononuclear infiltrate on 1
female – – also on
untreated skin
Skin biopsies: mononuclear
infiltrates
Not performed
10
Hanford
10
Hanford
Normal
12
Hanford
10%
Mild dose-site granulomatous infiltrate
10
Hanford
20%
Chronic dermis inflammation at dose site
10
Hanford
Well-tolerated
0%
Mild erythema and edema
beginning after 40 days,
slight to severe papules
and pustules beginning
after day 30
Mild edema pre- and postdose 2 separate
occasions
One male with persistent
erythema/edema, then
miliary erythema and
exudate, one female with
slight to moderate miliary erythema beginning
day 7
Mild persistent irritation
50%
and 83%
10%
Normal
10
Hanford
Intermittent dose-site erythema, varied occurrence
and severity
50%
Mild superficial dermis
inflammation
10
Hanford
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8
A. Stricker-Krongrad et al.
response during treatment or histologic pathology include a
combination of dehydrated ethanol, hexylene glycol, dimethiconol blend 20, hydroxypropylcellulose and anhydrous citric
acid. Methyparaben and propylparaben were also welltolerated. Water, polawax, denatured alcohol, propylene
glycol, isopropyl myristate, sodium hydroxide solution,
phenoxyethanol and carbomer 974P was a combination that
was well-tolerated for 210 days, but adequate comparison to
the other vehicles is not possible since histopathology was not
performed. Other vehicles resulted in mild, transient
responses. For example, a 50:50 composition of ethanol and
propylene glycol with very small amounts of BHA and BHT
produced very mild erythema in eight of the 12 animals on the
first day of treatment, but this was resolved by the next
observation seven days later in all but one animal. A vehicle
of soybean oil, coconut oil, mineral oil, cyclomethicone,
cetostearyl alcohol, stearic acid, myristyl alcohol, beeswax,
stearyl alcohol, and docosanol resulted in mild pre- and postdose edema in one animal on two separate occasions, and
mild granulomatous infiltrate in one of 10 animals on
histopathology. A vehicle made of gelatin phosphate buffer
resulted in slight erythema in one of 10 animals on day 2 that
was resolved by day 3. This particular study analyzed periodic
dose-site punch biopsies; three of the animals had mononuclear infiltrates on histopathology from various days
throughout the study.
Other vehicles were observed to cause decidedly negative
side effects. A vehicle of purified water, sodium chloride,
potassium chloride, L-arginine, glyceryl stearate, cetyl alcohol,
propylene glycol, squalene, polysorbate 20, sodium hydroxide
solution, oleic acid, isopropyl myristate, and keltrol RD and
BT resulted in severe erythema in two animals that began on
day two and persisted throughout treatment; histopathology
revealed these animals had developed bacterial infections as
well. PEG 400 caused adverse effects after 30 days of
treatment in over half of the animals; these effects consisted of
erythema and edema and slight to moderate, occasionally
severe, papules and pustules. Interestingly enough, histopathology revealed no abnormal findings.
Summary
Swine as a model in toxicity testing of pharmaceuticals and
other chemicals is now being well-accepted by Japan, EU
and USA regulatory agencies. Swine are specifically
mentioned as a potential non-rodent species in the guidelines of Japan and Canada and would generally be
considered superior to dogs and rabbits as a dermal
model. The OECD 409 guideline lists swine and minipigs
as optional species. However, evidence should be provided
that it is a suitable species in order to overcome residual
regulatory resistance. Increases in the amount of background information on this species will continue to
demonstrate their usefulness in toxicology in general and
specifically as a dermal model2,3,6,11,24,34.
Declaration of interest
The authors report no conflicts of interest.
Cutan Ocul Toxicol, Early Online: 1–9
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