United States
Environmental Protection
Agency f
Pesticides And
Toxic Substances
(TS-796)
20T-2004
August 1990
v°/EPA Atlas Of Dermal Lesions
REGION VI LIBRARY
U.S, ENVIRONMENTAL PROTECTION
AGENCY
1445 ROSS AVENUE •?
OAltAS, TEXAS 7520?
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United States Pesticides And 20T-2004
Environmental Protection Toxic Substances August 1990
Agency (TS-796)
&EPA Atlas Of Dermal Lesions
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PREFACE
This atlas was developed after broad consultation with experts in pathology and toxicology
from academe, industry and various government agencies. Readers seeking more background
information can go to the references cited on page 2. EPA scientists who contributed to the
preparation of this atlas included Drs. Mary Argus, Karl Baetcke, Diane Beal, James Murphy,
Stephen Nesnow, Vanessa Vu, and Mrs. Vivian Turner Williams.
EPA was assisted in this effort by the Dynamac Corporation, under contract number
68-01-7266, and by Clement International, Inc., under contract number 68-01-7290.
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TABLE OF CONTENTS
INTRODUCTION 1
Fig. 1 - Acanthosis, parakeratosis, and hyperkeratosis 3
Fig. 2 - Minimal focal epidermal necrosis 3
Fig. 3 - Minimal focal epidermal necrosis 4
Fig. 4 - Focal epidermal necrosis, hyperkeratosis, parakeratosis, and
acute dermal inflammation 4
Fig. 5 - Multifocal epidermal necrosis and ulceration 5
Fig. 6 - Focal epidermal necrosis and ulceration 5
Fig. 7 - Extensive necrosis, ulceration, and crust formation 6
Fig. 8 - Focal crateriform epidermal ulceration 6
Fig. 9 - Subcorneal microvescicles 7
Fig. 10 - Intraepithelial pustule 7
Fig. 11 - Intraepithelial pustule 8
Fig. 12 - Intraepithelial pustule filled with neutrophils and
serous fluid 8
Fig. 13 - Subepidermal pustule 9
Fig. 14 - Subepidermal pustule (dermoepidermal junction) 9
Fig. 15 - Focal epidermal spongiosis 10
Fig. 16 - Epidermal spongiosis 10
Fig. 17 - Hydropic degenerative change in the basal layer and
lower stratum spinosum of the epidermis 11
Fig. 18 - Intracellular edema 11
Fig. 19 - Dyskeratotic cells from basal layer to the
mid-stratum spinosum 12
Fig. 20 - Exocytosis 12
Fig. 21 - Dermal fibroplasia 13
Fig. 22 - Dermal fibroplasia, lichenoid distribution 13
Fig. 23 - Dermal fibroplasia associated with mild inflammation
and hemorrhage 14
Fig. 24 - Dermal edema 14
Fig. 25 - Epidermal ulceration, crust formation, and marked
focal dermal inflammation 15
Fig. 26 - Epidermal ulceration, crust formation, and marked
focal dermal inflammation (higher magnification) 15
Fig. 27 - Hyperplasia and atrophy of sebaceous glands 16
Fig. 28 - Hyperplasia of sebaceous glands 16
Fig. 29 - Dysplasia 17
Fig. 30 - Dysplasia 17
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INTRODUCTION
In April 1987, the Health and Environmental Review Division (HERD), Office of Toxic
Substances, conducted an EPA Workshop to discuss issues involved in (a) the design of a bioassay
protocol for chemicals by the dermal route, and (b) the feasibility of a screening assay for the
potential oncogenicity of acrylates/methacrylates (EPA 1989a). As a result of that workshop, the
issue of using histopathologic evidence of irritation to help determine the level at which the
integrity of the skin is destroyed [possible indicator of the maximum tolerated dose (MTD)] was
identified as an area requiring further analysis prior to the development of a dermal bioassay
protocol. A second workshop was held in Research Triangle Park, NC in May 1988 to address the
feasibility of formulating a system which would serve as a guideline to aid pathologists in arriving
at consistent diagnosis/grading of skin lesions to be used in establishing an MTD for chronic studies
(EPA 1989b). A set of proposed guidelines was sent out for review by both pathologists and
toxicologists. As a result of the responses received and the discussions at the RTP Workshop, there
was agreement on the gross and histologic conditions which constitute criteria for having reached
or having exceeded the MTD.
In conjunction with the grading guidelines, a set of photomicrographs of dermal lesions
considered to be representative of the spectrum of dermal irritation/toxicity reactions seen in
dermal studies was reviewed by a group of pathologists at the RTP Workshop. The
photomicrographs were selected from prechronic "skin paint" studies conducted by the National
Toxicology Program (NTP).
HERD requested the Experimental Pathology Branch of the NTP to provide information
regarding "skin paint" studies performed under the direction of the NTP. The NTP was selected
because of its well-recognized international stature in the field of experimental pathology and
expertise in "state-of-the-art" toxicology/pathology studies in rodents. The wealth of archival
material maintained by the NTP from approximately 23 subchronic and/or chronic "skin painting"
on rats and/or mice was also considered to be an important source of data available for review.
The NTP recommended review of recent prechronic "skin paint" studies which were available
at the NTP Archives. A site visit was made to the archives on September 24th and 25th, 1987, for
the purpose of examining tissue sections from rodent "skin paint" studies. Random microscopic
evaluation of skin sections from high does groups was carried out in rats and mice from
benzethonium chloride, triethanolamine and vinylcyclohexene diepoxide prechronic studies.
Review was limited to high dose groups because these groups appeared to manifest the greatest
variety of changes ranging from thickening to frank ulceration. The microscopic review was
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conducted to detect the presence of certain lesions characteristic of skin toxicity reactions. A
spectrum of lesions typical of changes frequently found in skin irritation studies was present in this
archival material. It was from this material that the study set of representative lesions was selected.
The object of the pathology segment of the RTF Workshop was to generate a working guideline
for use by pathologists in establishing an MTD for conducting chemical exposure studies by the
cutaneous route. This was to be accomplished by demonstration of lesions that occur commonly
during the performance of "skin paint" studies and by achieving a consensus regarding the
diagnostic terminology used to describe the lesions.
As futher background to the development of guidelines for a dermal bioassay protocol, the
pathology panel was also charged with defining what lesions constitute a compromise in the
integrity of the skin, arriving at a consensus regarding lesions that should and should not be
permitted for the MTD, discussing the feasibility of lesions grading/scoring, and recommending the
appropriate method(s) for handling skin lesions at necropsy to promote uniform and consistent
sampling.
SUMMARY
A consensus was reached by the panel of pathologists at the RTF Workshop regarding the
diagnostic terminology to be used for the microscopic lesions that were presented. It was agreed
that the lesions presented represent the most common changes that would be encountered in a
dermal toxicity study. The panel stressed that a thorough description of all lesions present in the
photomicrographs, as well as the major lesions, should be included in the guidelines. It was
recommended that the anatomical extent of the reaction should be indicated as well as whether it is
focal or diffuse in distribution (e.g., perifollicular, epidermal, dermal, subcutaneous, etc.).
The panel of pathologists emphasized that the magnitude of the gross lesions should be
correlated with the microscopic changes and that microscopy should be used as confirmatory
evidence for the grossly observed changes.
REFERENCES
EPA 1989a. Environmental Protection Agency: "Summary of the EPA Workshop on Carcinogenesis Bioassay via the
Dermal Route, April 28-29,1987, Washington, DC". (EPA 560/6-89-002); available from NTIS, 5284 Port Royal
Road, Springfield, VA 22161 (703 487-4650).
EPA 1989b. Environmental Protection Agency: "Summary of the Second EPA Workshopon Carcinogenesis Bioassay
via the Dermal Route, May 18-19,1988, Research Triangle Park, NC". (EPA 560/6-89-003); available from NTIS,
5284 Port Royal Road, Springfield, VA 22161 (703 487-4650).
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Fig. 1.
Hg. 2.
Acanthosis, parakeratosis, and hyperkeratosis. A mild inflammatory cellular infiltrate
is present in the dermis. benzethonium chloride. 25 mg/kg, ethanol vehicle. Rat. x33
Minimal focal epidermal necrosis. Acanthosis and hyperkeratosis are present at the
junction of the necrotic and viable epidermis. A diffuse moderate, suppurative
inflammatory infiltrate is present in the dermis. benzethonium chloride. 25 mg/kg,
ethanol vehicle. Rat. x33
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Fig. 3.
Fig. 4.
Minimal focal epidermal necrosis. The viable epidermis is acanthotic and
hyperkeratosis is evident. Focal epidermal ulceration is noted at the left. The dermis is
mildly and diffusely infiltrated with inflammatory cells, benzethonium chloride. 25
mg/kg, ethanol vehicle. Rat. x50
Focal epidermal necrosis, hyperkeratosis, parakeratosis, and acute dermal
inflammation. The adjacent viable epidermis is mildly acantotic and hyperkeratotic.
benzethonium chloride. 25 mg/kg, ethanol vehicle. Rat. x50
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Fig. 5.
Fig. 6.
Multifocal epidermal necrosis and ulceration. A thick crust is present over the affected
epidermis, benzethonium chloride. 25 mg/kg, ethanol vehicle. Rat. x!3.2
Focal epidermal necrosis and ulceration. A crust of necrotic epithelium, erythrocytes,
serum, and inflammatory cells covers the focal ulcer. The epidermis at the left is
acanthotic. benzethonium chloride. 25 mg/kg, ethanol vehicle. Rat. 50
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Fig. 7.
Fig. 8.
Extensive necrosis, ulceration and crust formation. The dermis and subcutis are
markedly infiltrated with inflammatory cells to the level of the panniculus. The
epidermis at the edge of the ulcer (at the right) is acanthotic and hyperkeratotic.
benzethonium chloride. 25 mg/kg, ethanol vehicle. Rat. x!3.2
Focal crateriform epidermal ulceration. The ulcer is covered by a crust and the
underlying dermal collagen shows basophilic degenerative change. Acanthosis is
evident at the edges of the ulcer. Moderate inflammation extends from the base of the
ulcer into the subcutis. benzethonium chloride. 25 mg/kg, ethanol vehicle. Rat. x!6
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Fig. 9.
Subcorneal microvesicles. Hydropic degenerative change is noted in the epithelium
beneath the microvesicles. The epidermis is acanthotic and hyperkeratotic.
vinylcyclohexene diepoxide. 100 mg/ml, acetone vehicle. Rat. xlOO
Fig. 10.
Intraepithelial pustule (microabscess) of perifollicular epithelium. Mild exocytosis of
acute inflammatory cells is present in the surrounding epithelium, vinylcyclohexene
diepoxide. 100 mg/ml, acetone vehicle. Rat. x40.
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Fig. 11.
Fig. 12.
Intraepithelial pustule. The pustule contains serous fluid, erythrocytes and numerous
polymorphonuclear leukocytes. The "roof of the pustule to the left appears ruptured.
The viable epidermis to the right is acanthotic, the superficial dermis is edematous and
has a mild inflammatory cellular infiltrate, vinylcyclohexene diepoxide. 100 mg/ml,
acetone vehicle. Rat. xlOO
Intraepithelial pustule filled with neutrophils and serous fluid. The epidermis is
acanthotic with prominent exocytosis. Superficial dermal inflammation is evident.
vinylcyclohexene diepoxide. 100 mg/ml, acetone vehicle. Rat. x50
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Fig. 13. Subepidermal pustule. The inflammatory cells are located at the dermoepidermal
junction and the covering epidermis is necrotic. vinylcyclohexene diepoxide. 100
mg/ml, acetone vehicle. Rat. x50
Fig. 14. Subepidermal pustule (dermoepidermal junction). The pustule is covered by necrotic
epidermis and suppurative inflammation is evident in the dermis beneath the pustule.
Acanthosis, hyperkeratosis, and parakeratosis are present in the adjacent epidermis.
vinylcyclohexene diepoxide. 100 mg/ml, acetone vehicle. Rat. x50
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Fig. 15.
Fig. 16.
Focal epidermal spongiosis. Note the widening of the intercellular spaces in the stratum
spinosum and edema of the superficial dermis beneath the spongiotic area. Acanthosis
and hyperkeratosis are evident in the epithelium and crust formation is noted at the right.
A mild dermal inflammatory response is evident, benzethonium chloride. 25 mg/kg,
ethanol vehicle. Rat. x33
r
Epidermal spongiosis. Widening of intercellular spaces is present in the basal layer as
well as the stratum spinosum. Coalescence of areas of intercellular edema suggest early
microvesicle formation. The underlying dermis is edematous and shows dilatation of
capillaries and small lymphatic vessels. A single eosinophilic dyskeratotic cell is
observed at the mid-level of the stratum spinosum. triethanolamine. 2 gm/kg, acetone
vehicle. Rat. x80
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Fig. 17.
Hydropic degenerative change in the basal layer and lower stratum spinosum of the
epidermis. Mild hyperkeratosis is present. Mild superficial dermal inflammation is
evident, vinylcyclohexene diepoxide. 100 mg/ml, acetone vehicle. Rat. x50
Fig. 18.
Intracellular edema. Epithelial cells of the basal and stratum spinosum layers have
swollen pale water-clear cytoplasm with eccentric pyknotic nuclei. Single necrotic
cells (eosinophilic and shrunken) are noted at the left. Hyperkeratosis is present.
vinylcyclohexene diepoxide. 100 mg/ml, acetone vehicle. Rat. x80
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Fig. 19.
Dyskeratotic cells are noted from basal layer to the mid-stratum spinosum. -The
cytoplasm is eosinophilic and abundant and is occurring in the non-keratinizing area of
the epidermis. The epidermis is acanthotic and hyperkeratotic. vinylcyclohexene
diepoxide. 100 mg/ml, acetone vehicle. Rat. x80
Fig. 20.
Exocytosis. Suppurative inflammatory cells are migrating through the acanthotic
epidermis. A similar inflammatory response is noted in the superficial dermis.
vinylcyclohexene diepoxide. 100 mg/ml, acetone vehicle. Mouse. x80
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Fig. 21. Dermal fibroplasia. Immature fibrocytes proliferating in a lichenoid pattern beneath the
acanthotic, hyperkeratotic epidermis, triethanolamine. 4 gm/kg, acetone vehicle.
Mouse. x40
Fig. 22. Dermal fibroplasia, lichenoid distribution. Marked acanthosis with mild hyperkeratosis
is evident, triethanolamine. 4 gm/kg, acetone vehicle. Mouse. x!6
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Fig. 23.
Dermal fibroplasia associated with mild inflammation and hemorrhage. The epidermis
is moderately acanthotic and hyperkeratotic. triethanolamine. 4 gm/kg, acetone
vehicle. Mouse. x40
Fig. 24.
Dermal edema. A pale staining zone of interstitial edema is evident in the superficial
dermis. Dilatation of small lymphatic vessels is noted in this area. The epidermis shows
marked acanthosis with rete peg formation at the left. A large unattached crust is above
the epidermis, vinylcyclohexene chloride. 100 mg/ml, acetone vehicle. Rat. x40
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Fig. 25.
Epidermal ulceration, crust formation and marked, focal dermal inflammation. The
inflammatory reaction extends into the subcutis and the overlying epidermis shows
acanthosis and hyperkeratosis. benzethonium chloride. 25 mg/kg, ethanol vehicle. Rat.
x!3.2
Fig. 26.
Higher magnification of Fig. 25. Epidermal ulcer with attached crust is at the right,
acanthotic hyperkeratotic epidermis is at the left, and a marked chronic, active
inflammatory response is present in the dermis. benzethonium chloride. 25 mg/kg,
ethanol vehicle. Rat. xlOO
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Fig. 27.
Hyperplasia (at right) and atrophy (at left) of sebaceous glands. The atrophied glands lie
beneath an area of extensive ulceration; the hyperplastic glands beneath acanthotic and
hyperkeratotic epidermis, benzethonium chloride. 25 mg/kg, ethanol vehicle. Rat. x33
Fig. 28.
Hyperplasia of sebaceous glands. Acanthosis, hyperkeratosis, crust formation and a
small focal ulcer are seen in the epidermis. Mild dermal inflammation is also present.
benzethonium chloride. 25 mg/kg, ethanol vehicle. Rat. 33
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Fig. 29.
Dysplasia. Focally disorganized growth pattern of epithelial cells. Loss of polarity
(lack of stratified growth) and mild nuclear atypia characterize this change. Acanthosis
and hyperkeratosis of adjacent epidermis is evident, vinylcyclohexene diepoxide. 100
mg/ml, acetone vehicle. Mouse. x50
Fig. 30.
Dysplasia. Faulty stratification of the epidermis with nuclear atypia. Keratinization is
abrupt above this disorganized area of epidermal growth. Moderate hyperkeratosis and
minimal parakeratosis are noted, vinylcyclohexene diepoxide. 100 mg/ml, acetone
vehicle. Mouse. x60
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