EPA560/6-89-003
Summary of Che Second EPA Workshop on Carci.no genes is Btoassav
via the Dermal Route
Mav 18 • 19. 1988
Research Triangle Park. NC
EPA PARTICIPANT'S:
Dx. Mary Argus (Chairman**
Or. Karl Baatcka*
Ms. Vivian Turntt
Ms. Linda Cullan
OYHAMAC COtPOmtlOH;
Dr. Sh«ron A. Sagal*
TOXICOLOCT fAKEL:
Or. John Clary (fornarly with Calanasa Corp)*
Dr. Linval DtPass (Syntax Rasaarch)*
Dr. Skip Eastin (NIEHS/NTP)*
Dr. Jamas Fraanan (Exxon Blooadlcal Sciancas)
Dr. Skip Matthawa (NIEHS/NTF)
Dr. Jar.as HcOannof 'Proctar and Gambia)*
Dr. Staphan Naanoo (USEPA/HUU.!*
Or. J:n< Rlviara (NC Staia Unlv; School of Vat. Mad.)
Dr. Andruw Sivak (Arthur 0. Llttla. Inc.)*
PATBOLOGY PANP.;
Or. Ronald Schualar (Chairman * Dynanac. Consultant to EPA)
Dr. Michaal Elwall (NIEHS/NTP)
Dr. Ed Fowltr (Bushy Run Rasaarch Cantar)
Dr. Mi:haal Holland (Upjohn Co.)
Or. Gary Johnson (Proctar and Gambia)*
Dr. Andva J.P. Klaln-Szanto (Fox Chasa Cancar Cantar)
Dr. Joal Lalnlngar (NIEHS/HTP)
•Also particlpatad In tha 1st EPA Darnal Workshop (April 28-29, 1987)
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50377-1QI
REPORT DOCUMENTATION i «..««3«r no.
PAGE ! EPA560/6-89-003
PB90-K.6358
4. THIa and Subtitla
Soimary of the Second EPA Workshop on Carcinogenesis
Bioassay via the Dermal Route
Oncology Branch/HERD/OTS
9. Report 0*t •
Hay 18-19, 1988
9, Parforminf Orfanliation Rapt. No
V. Performing Organiialion Nama and Addrata
Dynamac Corporation
11140 Rockville Pike
Rockville, MD 20852
10. Proiact/Taak/Work Unit No.
103
II. ContracKC) or Qrant(C) No
(068-01-7266
12. Sponsoring Orfanitation Nama and Addratt
Office of Toxic Substances
US Environmental Protection Agency
401 M. Street, S.W.
Washington, DC 20460
:i. Tyoa of Raoon I Panod Covarad
f" inal Technica 1_ Repor t
14.
IS. Supplamantiry Notat
1*. AbatrKt (Limit: MO »ordl)
The purpose of this workshop was to gather information and expert
opinions to aid EPA in drawing up guidelines for carrying out
rarcinogenesis bioassays via the dermal route. At the first workshop in
April 1987, consensus was reached on may aspects of this problem. The
following areas were pinpointed as requiring further consideration in a
second workshop before the desired guidelines can be written:
1. the selection of species and strain
2. the role of pharmacokinetics
3. the establishing of the MTD (This includes the unique situation
where skin may be the target organ for the toxicity end-point to
arrive at the MTD).
At the April 1987 workshop it was decided that these end-points
involving the skin should be determined on both gross and histological
considerations, and that the second workshop should include both
pathologists and toxicologists. These considerations thus provide the
basis for the agenda and roster of participants for the second workshop.
17. Docurmmt Afialytll •• DoKrtptor*
Dermal Bioassay; Carcinogenesis; Skin Irritation
b. k»a««>«Of./Opa« end** Torm»
Dermal Bioassay Protocol; species/strain selection for dermal 9ncogenicity
(Maxirtun Tolerated Dose); histopathologic evidence of skin irritation; gro
testing; KID
gross criteria
for skin irritation
e. COSATI FMaVOrmp
It, Availability KaMmam
Docunent is available to the Public through the
National Technical Information Service; Springfield
VA 22151; unlimited release
If. Jacurrr, Cla» (TMa Mapoit)
. StcurHy CUM (Tklt ftft}
U. No. of Pa«M
12
(*4oANSI-Zn.lt|
I 272 (4-77)
(•armorr? NTIS-JS)
Poxitiimil •> C«iun*rc*
-------
The purpose of this workshop was to gather information and expert
opinions to aid EPA in drawing up guidelines for carrying out carcinogenesis
btoassays via the dermal route. At the first workshop in April 1987 consensus
was reached on many aspects of this problem. The following areas were
pinpointed as requiring further consideration in a second workshop before the
desired guidelines can be writted.
1 the selection of species and strain
2 the role of pharmacokinetics
3 the establishing of the l CD (This includes the unique situation
where skin may be the target organ for the toxicity end-point to
arrive at the MTD).
At the April 1981 workshop it was decided that these end-points
involving the skin should be determined on both gross and histologicaL
considerations, and that the second workshop should include both pathologists
and toxicologists These considerations thus provide the basis for the agenda
and roster of participants for the second workshop
I SPECtES/STR.AIN SELECTION
Introduction
As a result of the first EPA Workshop on the design of a protocol for lor.g-
term tisting by the derinal route, the selection of the most suitable test
animals was identified as a subject requiring further evaluation The moice
was suggested as one appropriate test species, but it was decided that the
selection of an appro riate mouse strain and a second test species (the mos
likely candidates being the rat or the hamster) could not be made until the
available data base on the use of these animals in dermal studies had been
reviewed As a result of reviewing that data base 1 a document was prepared
that defined the scope and asseszed the quality of information available on
the use of mice, rats, and hamsters in dertual carcinogenicity studies and
presented an analysis of the findings to be used to help identify appropriate
rodent species and strains for use in bioassay testing by the dermal route
This section presents a su ary of the discussions concerning this issue
Mouse
The dermal protocol should not be limited to any one specific strain or stock
of mice
The existing data base indicates that seven strains or stocks of mice (BALB/c,
B6C3F1, CD-l, C3H, C5751/6, Swiss (ICR) and SENCAR) are susceptible to
tumorigenesis by the dermal route. While the Workgroup recommended the use of
these strainsjstocks in a derma] bioassay, the experimenter is not limited to
the use of only these strains if there is justification.
It was noted that problems with genetic drift and the lack of availability of
the SENCAR mouse may reduce its suitability as a test strain.
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It was pointed out that the skh/hr (hairless) mouse has been used to study
photocarcinogenesis by the cutaneous (dermat) route
The experimenter must justify the choice of strain/stock of mice used for the
testing of a given chemical (see criteria below)
The existing data base is limited and does not indicate that any single strain
of rat is more appropriate to: dermal bioassay testing than another
The 1144 rat is the recommended strain for use in a dermal bioassay, based on
the availability of a well-developed existing NT? spontaneous tumor data base
and an anticipated dermal data base However, the dermal protocol is not
limited to the use of this strain if there is justification Co use another
one
The experimenter must justify the choice of strain of rat used for the testing
of a given chemical (see criteria below).
Criteria for Selection of Mouse and Rat StratniStock
The following criteria (in approximate order of importance) should be included
in the justification of the choice of rocLut strain/stock to be used
1 Susceptibility of the strain to the class of chemicals
to which the cest chemical belongs
2 Incidence of spontaneous tumors that may compromise
skin study (a g C3H mice/mammary tumors)
3 Survivabilsty
4 Adequate historical data base
S Resistance to irritation
6 Availability/breeder specificity/genetic stability for
inbred strains (consistent response)
Syrian Colden Hamster
The available data base, which is limited , indicates that the Syrian golden
hamster may be more susceptible than the rat to the development of both dental
and systemic tumors following dernal exposure. However, hamster husbandry is
difficult, sutvivability is considered low, and there is a paucity of data on
dermal absorption in hamsters.
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Rat Vericas Hamster as a Second Soecies
While the rat is not as susceptible as the Syrian golden hamster to the
development of tumors at the sits of application (skin), IrritatLon and derraal
absorption/pharmacokinetics have been more extensively studied in rats
Dermal application of carcinogens induces primarily basal cell carcinomas in
rats and squamous cell carcinomas and melanomas’in hamsters
Based on the incidences in humans of the different types of malignant tumors
of the skin, the types of skin tumors that develop in rats (basal cell
carcinomas) were considered more relevant than those observed in hamsters to
human skin carcinogenesis.
Conc lus ions
The mouse was recommended as the first test species based on its greater
susceptibility to skin carcinogenesis (when rat responds, generally much
higher doses are required), and the rat should be selected as the second test
species based on the availability of extensive systemic carctnogsnicity
studies and absorption/pharmacokinetic data, as well as the existence of an
established historical control data bass.
Special circumstances may provide justification for (1) the use of the hamster
as the second species, (2) conducting a dermal study in mice in conjunction
with an alternate route (e.g. oral) in rats or; (3) conducting dermal studies
in two strains of mice.
I I PHARNACOKINE IICS/ABSORPTION DATA FOR DERMAL BIOASSAY
Introduction
One of the issues discussed at the first EPA Workshop on the design of a
dermal bioassay protocol, concerned the acquisition of
absorption/pharmacokinetic data, To summarize
“Regardless of whether the toxicity endpoint for estimating the
MTD involves the skin or is systemic, it was agreed that a 90-day
dose-finding study is necessary to determine an MTD for the long-
term bioassay. Short-ten toxicity, absor tionL oharmacokinetic
data shouLd be available orior to conductinç the 90-day _ dose-
finding study Microscopic examination of the skin should be part
of the 90-day study. Skin absorotion studies also should be done
at several time ooints durin,g. _ çhe study and at the end of the 90
days to provide data on differences in absorotion with time durina
exposure .
In addition to dose selection, it was also felt that
absorption/phartiacokinetic data would aid in determining if a particular
chemical can be adequately tested by the denal route.
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While the importance of gathering absorption data at various time intervals
was recognized at the first EPA Workshop, no attempt was made at that time to
characterize the type and extent of these studies To do so. the following
questions become apparent and were raised to the second EPA Workshop
participants
1) What are the minimum data that are considered
acceptable for determining whether a chemical is
absorbed following dermal application? Is it possible
that blood level detection (of a chemical) is
suffLcLent to indicate absorption?
2) What pharmacokinetic/absorption data are most useful
in determining an MTD for dermal studies?
3) What is the role of j vitro data?
Discussion
l What are :he minimum data that are considered acceptable for deterlninLng
whether a chemical is absorbed following dermal application? Is it possible
that blood level detection (of a chemical) is sufficient to indicate
absorp t ion?
The lack of detection of a chemical in the blood is not sufficient criteria
for concluding non-absorption of chat chemical. Failure to detect the
chemical in the blood could be due to other factors (e.g. time of measurement,
rapid clearance, analytical sensitivity, accumulation in certain organs)
Although pharmacokinetic data are not essential to the determination of
carcinogenic potential, such data are important to a comprehensive
interpretation of the bioassay data. Thus, it is preferable to know to which
organs a chemical is distributed, as well as the chemical specTies chat are
absorbed One suggested method for determining absorption and distribution
was the use of a radioactive compound in a whole body autoradiography study
(This method provides only an indication of chemical distribution) The
“balance study , which allows one to determine what is expired and/or
excreted, was the most widely accepted example of a procedure which would
provide the minimally acceptable data for judging absorption. Following is a
composite balance study, incorporating the important points that were
discussed.
A single dose of radiolabelled chemical (specific activity sufficient to
determine 0.1% absorption) is applied to the skin of 3 animals/dose, 3
doses. The high dose is approximately 10% of the LD, 0 (oral, or dermal
if avai4able, or as high as can be reasonably applied to a 1 to 2 cm 2
area of skin). The low dose should not be less than the detection
limits or human exposure (if known). The mid dose should ideally fall
one log increment above the low dose unless there are less than two or
more log increments between the high and low doses, in which case it is
midway between the high and low doses. A nonocc lu sive stainless steel
mesh cup is affixed over the application sita so that the animal cannot
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ingest the material, but that the chemical is exposed to the environment
(to mimic the conditions of the chronic study). The animal is put into
a glass metabolism cage and volatiles and expired carbon dioxide, urine
and feces are collected for 72 hours. The level of radioactivity is
determined in the volatiles and eliminated material, blood, site of
application (skin), certain selected organs and remaining carcass An
acceptable level of recovery is between 90 and 110% of the radioactivity
administered
Information obtained from the balance study, conducted as described, can only
be used qualitatively for extrapolation to humans
The same procedure may be conducted simultaneously in animals that have been
injected intravenously as a means of obtaining data representing 100%
absorption In conjunction with the intravenous data, the balance study
provides a quantitative estimate of absorpt.ion of the test chemical from a
given skin area in the test species and the effect of a dose on absorption
These data, however, provide only a qualitative estimate of absorption in
other test species or humans.
The following issues were also discussed:
o The summa f the first Workshop (see above) indtcaLe that
pharmacokinecic/absorption data should be obtained at multiple time
points throughout the 90-day study. One of these intervals should be at
the time that any evidence of dermal irritation has subsided.
o Should a “hetical be tested for its inherent carcinogenicity, regardless
of the route of exposure or should its carcinogenic potential be studied
only by the route of expected human exposure (i e. dental)? For the
former, absorption data would play a significant role in determining the
route of exposure for the bioassay, but for the latter, results of
absorption studies would be irrelevant, If the potential route of human
exposure is dermal, then even if absorption studies indicate no
measurable absorption in a second species, a denial study would still be
conducted in the mouse, since the possibility of local skin effects and
skin tumor development without apparent systemic bioavailability cannot
be ruled out. Absorption data would be used to determine the route of
administration for the second species, the rat, in most cases. If there
is little or no systemic absorption after dermal application to this
second species, then another route that provides greater systemic
exposure should be used for the second study. Thus, it was concluded
that the absorption/pharmacokinetic studies will be carried out only in
the second species (rat or hamster) and will not be necessary for the
mouse. —
o The tens penetration’ and absorption are not interchangeable. While
skin penetration is necessary for skin tumonigenesis, absorption is
required for systemic bioavailability.
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o The option was also discussed for not evaluating all organs
histologically, if no absorption is fc.’und over 90 days. However, it was
discussed and agreed that for both humans and experimtntal animals,
ingestion of a chemical is always present to a certain degree in dermal
exposure situations.
2. What pharniacokinetic/absorption data are most useful in determining an
MTD for dermal studies?
It was agreed that classical systemic toxicity enopoints and/or the gross or
histologic skin toxicity endpoints concurred on at this meeting should be used
in determining an L CD for dermal studies and that pharmacokinetic/absorption
studies are not necessary; they are useful in the interpretation of the final
data.
3. What is the role of ft vitro data?
j vitro data give only a perspective on species comparisons (e.g. between
first and second species and especially if no absorption is detected in the
second species). The data base in this area is currently lacking for
comparing ft vitro to ft vivo absorption. j vitro data are useful for
vehicle selection and helpful in dose selection for range.finding studies
III. PATHOLOGY EVALUATION
Introduction
The issue of using histopathologic . evidence of irritation to help determine
the level at which the integrity of the skin is destroyed (possible incicator
of MTD) was identified as an area requiring further analysis prior to the
development of a dermal bioassay protocol at the first EPA Workshop.
Toward this goal, we investigated the feasibility of formulating a system
which would serve as a tuideline to aid pathologists in arriving at consistent
diagnosis/grading of skin lesions to be used in establishing an MTD for
chronic studies. 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 agreetaent on the gross and
histologic conditions which constitute criteria for having reached or having
exceeded the MTD.
Evaluation of Photomicrog jphs
o A high level of agreement was reached on the diagnostic aspects of the
microscopic lesions. It was generally agreed that the changes represent
the most coon lesions that would be encountered in a dermal toxicity
study. A thorough description of all lesions present in the
photomicrographs, in addition to the major lesions, was stressed. It
was suggested that the anatomical location of the reaction should be
indicated, as well as whether it is focal or diffuse in distribution
(e.g. perifollicular, epidermal, etc.).
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o It was stressed that the magnitude of the gross lesions should be
correlated with the microscopic lesions, and that microscopy will often
be used as a backup to confirm the gross changes.
o It was further recommended that the term dysplasia” be used to include
atypia. anaplasia, and carcinoma-in-situ.
Effect of Pathologic Changes on Selection o [ MTD
o Prior to selection of doses f r the subchronic study, it was recommended
that a 2-week study bq done to detect dermal toxicity Application
should be daily. 7 days/week. A thorough evaluation of gross lesions at
termination would then be used to select the dose levels for the 90-day
subchronic study. A miriimu of 10 animals/sex/dose should be used in
the 90-day study.
o It was recommended that a dose level that incites a marked inflammatory
response or ulcerative lesion that is clearly related to application of
the compound, should not be used for an MID.
o It was emtehasized that chemical irritation must be distinguished from
complicating secondary effects of bacterial dermatitis, clipper cuts,
self•mutilation from scratching, etc.
o To ensure that the MID has been reached, consistent changes in a
majority (i.e. 6 of 10) of the animals must be present.
o Microscopic lesions of inflammation, spongiosis, degeneration, dermal
edema, and possibly others, must be evaluated carefully in the selection
of the MID. If, in the opinion of the pathologist, the severity of such
lesions miaht lead to destruction of the functional intetritv of the
eDidermis , these lesions would indicate selecting a lower dose for the
MID.
o Lesions such as epidermal hyperplasia, hyperkeratosis, parakeratosis,
dyskeratosis, dermal fibrosis, and atrophy/hyperplasia of adnexa are
permitted.
o Subjective grading of lesions (e.g. minimal, mild, moderate and marked)
should be left to the discretion of the individual pathologists The
subjective nature of grading has inherent weaknesses because of the
variable interpretations among different pathologists This, however,
does not preclude its use by individual pathologists for sorting out
lesion !everity.
o Animals with lesions that are judged to be marked should be euthanized
as soon as it is determined that the lesion is not going to heal.
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Recommendations for Necroosv
o Histologic evaluation is not necessary if zross evidence shows that the
MTD has been exceeded.
The proper preparation of skin sections requires tissues to be free of
artifacts and oriented to permit evaluations of epidervual, dermal and
folliculosebaceous units. Therefore:
o Sections of skin should be taken from the site of app 1 ication with
respett to the longitudinal axis of the animal and should include
subcutis and muscular layer for complete examination When oriented
with the longitudinal axis, follicular structures are better
demonstrated.
o Samples of skin taken at necropsy are flattened on a piece of
paperboard, gently stretched and adequately fixed in formalin prior to
trimming
o Step-sectioning every 50-75 microns will allow for full evaluation of
all skin structures
o. Photographs of gross lesions that are representative of the findings are
recommended for each dose level.
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PROPOSED MID _ GUIDELINES _ BASED ON DERHAL TOXICITY
CROSS CRITEP I A
For reaching MTD erythema (moderate)
scaling
edeta (mild)
alopecia
thickening
For exceeding l CD ulcers
fissures
exudate/crust (eschar)
non-viable (dead) tissue
HISTOLOGIC CRITERIA
For reaching l CD inflammation (moderate)
spongiosis (minimal to mild)
epidermal hyperplasia
hyperkeratos is/parakeratos is/
dyskeratosis/hyperplas ia
edema/fibrosis
atrophy/hyperplasia of adnexa
(hair follicles)
For exceeding lCD microulcers
spongiosis (moderate)
degeneration/necrosis (mild to
moderate)
crust formation (eschar)
inflaation (marked)
edema (marked)
anything leading to
destruction of the
functional integrity of the
epidermis (e.g. cracking.
fissuring 1 open sores
oschar)
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GLOSSARY • CROSS
Erythema reddened skin
Edema raised, swollen skin
Cracking superficial breaks
in the surface of
the skin
Fissuring deep cracks in the
skin, may be
accompanied by red
discharge or scabbing
Desquamation/Exfoliation loss of skin ranging from
small flakes to
sloughing of sheets or
larger areas of skin
(denuding)
Open Sore lesion on the skin
generally accompanied
by a discharge
Ulcer loss of epidermis
Eschar large areas of scab
formation
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GLOSSARY . MICROSrOPTr
Hyperkeratosis
increased thickness of the
stratum corneua
Parakeratosis
hyperkeratosis with retention
of nuclei
Hyperplasia/Acanthosis
increased thickness of non-
cornified epidermis due to
increased cell numbers
Spongiosis
intercellular edema
characterized by widening of
Intercellular spaces
Intracellular Edema
increase In cell size, pale*
staining cytoplasm and
eccentric nucleus
Ulcer
loss of epidermis with
exposure to the dermis
Erosion
superficial loss of epidermis
Crust Formation (Eschar)
a congealed aggregate of
keratin, serum, cellular
debris (inflammatory cells
and blood), microorganisms
Dyskeratosls
Metaplasia
premature keratlnization
change from one type of
differentiated tissue to
another type of
differentiated tissue
Dysplasia
abnormal development of the
epidermis including atypia.
anaplasla and carcinoma-in-
situ
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