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EPA560/6-89-002
mimarv of the EPA Workshop on Carcinogenesis Bioassay
via the Dermal Route
April- 28-29. 1987
Washingcon. D.C.
EPA PARTICI?A1ITS
Mary Ar
Karl. Sa
Diar.* 3
David
3ruct
Stephtr.
PYMAMM: CORPOKATIOB
^: . Sharon A. S«g«l
EXPERTS
r. John CUry (CeLar.«»« Corp.)
Ir. Llnval D«P«»« 'Synttx R«s««rch)
Dr. Wllllara E««tir. :SIESS;»TP)
Dr. Janph BHMian '.SIESS/MTP)
Dr. G«ry Johnion (Procttr «nd G«nbXt)
Cr Ralph Kod«ll
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SO21 101
R(PORT DOCUMENTATIO1’d 1. OIPORT 00 3. O D O01 A01•u o ,. No
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.rnnary of me EPA crkshop cr Carcirc enesi : iC5 via Anril 2 -i I
the DeCr2l Scute
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- Oncology Branch/HIBD/(YtS
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Dynamac crpcrat ion
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tfice of Tcxic SuostanCes
U.S. Erwi cnrnenta1 Protection Ayency Final Technical Repcrt
401 M. Street 1 5.W. 08/28—29/5 7
ashingtcn , DC 20460
IS. S005lOa ..01.ry Not.,
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Traditionally, the oral route has been the most common ro t .
of administration in bioassays which tested the potent ai
carcinogenicity of chemicals. Regulatory agencies, however
orefer to have test chemicals applied by the same route s
expected human exposure, whenever possible. Since human exposure
to indu tria1 chemicals is frequently via the dermal ro’te, this
has become a route of choice for animal testing of cert... n
chemicals. owever, protocol design far dermal bioassays
presents many unique problems which must be addressed before
juidei.ines for bioassays by the de mal route can be formulated.
Furthermore, tt may be feasible to develop a limited dermal
protocOl tO screen certain classes of chemicals such as
acryiates./methacry].ates. Recognizing the need for this workshop,
it was designed in two distinct parts; to address the problems
inherent in the development of (1) a generic protocol for dermal
hioassayS and, (2) a specific li”ited derrnal bioassay -rotocol
for acry lates/methacrylates.
17. Docoa’.nt Ao Iyo t .. DORC ,.pto ,i
Dermal Bioassay; Acrylates/rlethacrylates carcinogenesis
b. IdsMItt. ,,IQ OOR-Cnd,d t 5 ltlI
Identifiers/Opeflended terms
Dermal bioassay protocol; limited dermal protocol for screening acrylates/methacrylates.
skin irritation; M W
c. CO$A?I INIIG , op
LI. AoSItabI. 157 It tSl0*Ot IS. S,ooot CISRi CTN. . R.D0’tI 21. Ne. of P.$.4
Document is available to the public through the
Natic nal Technical Information Service, ——
Spririgfie1 , VA 22151; unlimited release 20. S.,I 1YCI.. Ifl.IOPOI.) . Po.o.
1$.. ANSh-Z3 5 .IS) SO. I001 ,vCtf000 OR 1000111 0T 10$tAf. 712 (&-77 1
{F.flOR ”l
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The need for ZrILS orkshop arose from the fact that Lit btoassavs to
test the potentLal carctnogenicity of chem i cals the oral route has been tne
most common route of administrat i on used Regulatory agencies however
prefer to have test chemicals applied by the same route as expected human
exposure whenever poss i ble S i nce human exposure to industrial chemicals is
freqta.ntt’. via the dermal route. th i s has beccme a route of choice for animal
testtng of certain checnLcals dowever protocol desLgn for dermal btoassays
prescnts many unique problems which must be addressed before guidelines for
5toassa s by the dermal route can be formulated Furthermore, it may be
feasible to develop a limited dermal protocol to screen certain classes of
,hemicals ..t’ere exposure is known to occur primarily via the dermal route
such as the acr’. lates/methacrvlates This workshop was designed in tvo
d sttnct parts to address the problems tnh .renc in the development of l a
&enenc protocol for dermal bioassay and (2) a specific limIted dermal
oLoassa’. protocol for acrvlates/methacrvlates
The oojectives ot the iorkshop :ere therefore to
lU define and address key issues invoLved in designing a protoco for
testing the carcinogenicity (both systemic and dermal) of
substances b. the dermal route
fl use the results of these discussions to explore the feasibil cv of
de”eioping a limited dermal protocol for :ne screening of
acrilates/methacrvlaces for oncogenic potenttal
E’.,erts ho partic..pated in the orksnop included John Car: Li ’ al
DePass .t.liam East in James McDermott Michael Moore Stephen ‘.esno ard
rdre S.’a who -era setected for the.t e per:ise in chemical arcLno&enesLs
.nch..dL”& s3ecial empnas..s on dermal or acr’.la:e carc nagenests and on
pnarwacoKirettcs Gary Johnson and Da’ i d Strayer, two oatho1og s:s
specLaL:L- ; in the skin and Raloh Kodell and Joseph aaseman, :_o
;:at.st.c:ans kno edgeable in e perimenta. des gn T’-e r::ten corrjpe :s of
:ese a terts in response to a questionraire aimed at .dentif’.ing and prob r’g
.ss es 1 d nobler’s related to the stated obiect.i es formed the hass for the
Lsc,ss rc at the 2 da’. orkshop
‘‘e onsens s of tre group stated at the outset as that aLterna:.’e
e oer me:a:Lon to animal testing should ai avs be cons i dered and zna:
Arrectssar replicat on should be avotded
Recom.’nendations for the Desian of a Protocol LQr a Caicjn _ ogenesis Bioassay via
the Dermal Route
Concerning Soecies an& Strain Selection
• It is most desirable to have :—o spec:es of artmal for der.nal
bioassay
• One soectes should be the mouse howe”er selection of the stra i n
of mouse can only he made after revtew of the existing data on the
reiat.ve susceptibih i t.. of different mouse strains to sk r
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irritatton and to tu mor induction following applicat i on of
chemicals to the skin
• Both tne rat and the hamster may be viable OPtLOflS for the second
species, however, careful analysis of the existing data on the
use to date, of these two species in carcinogenesis studies by
the dermal route is necessary before a decision can be made
• The rabbit and guirea pig were ruled out as feasible species for
dermal bioassay testing, mainly because of technical difficulties,
expense and lack of a historic database
• If after review of rhe existing databases on the rat and tne
1 iamster a second species can not be identified that is acceptable
for dermal bioassay tc’sting. then the possibility of testing n
: 1 -e rat b the oral route in conjunction with the dermal tes: ng
of cne mo’ se was suggei ted
• Another less desirable possibility suggested by the group was tnat
renew of the existing catabase or different strains of mice used
:n dermal testing may reveal that there is a difference between
mouse strains with respect to susceptibility to irritation, and
that th s could possibly be the basis for the selection of two
different strains of mouse to be used in dermaL bioassay tes::ng
rather :nan two different spec es
Dose ‘eLection
egardless cC -nether the toxicity endpoint for estimating : e “i’D
.n”o. ‘es the s i or L5 svstemtc. it -as agreed that a °O-oa.
dose flnd rtg stuc’ s necessary to determine an MID for the long
ten oLoassa ? Shnrt.teLm toxicity absorptLon,pharmacokinetic
,!a:a sho Id e .va,.labLe prior to conduc: ng the 90 das dose-
:rcrg s:ud: Microscopic examination of the skin should be part
f :‘e u da t study Skin absorption studies also should be done
it ce etut :Lme points during the study and a: the end of ;ne 90
ha s to 3ro%ide ca:a on differe’ices :n absorption w th time dur:ng
— ;osui’e
• .s irpera:t’e that the same test animal (species and strai-t) be
nec Lor the subchronic range-finding studies as fot the loi;-term
ioassav regardless of what species may have been used in an acute
or stzbcni-oriic irritation stud’: for the same chemca
• Thj Jrai:e s
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scoring method for visual and microscopic irritationpf the skin
• It as also agreed that when the skin response is used to
determine the MID, the desired endpoint is the highest dose hhere
the integrity of the skin is not destroyed A more explicit
definition is needed for dec i ding when the “integrity of the stun”
is destroyed, and this must also be decided by the above-mentioned
panel The consensus of the group was that hyperplasia and
h’.rperkeracosis is acceptable, that ulceration of the skin is
unacceptable, and that factors such as changes in the skin in
regards to flux, permeability, etc , as well as the reversibility
of any changes seen must be considered in est.mating an MID
• It as generally agreed that 3 dose levels (plus a control) be
used and the MTD should be the highest dose in the long term
bioassay When skin irritation is a factor, the ideal choice for
the ocher two doses would be one dose that is minimally irritating
and one that is the highest non- irritating dose Another basis
for choosing doses considers chemical-specific data (i e
absorption, kinetics, toxicity, etc ) and therefore it is
difficult to assigr an arbitrary and generic method for selecting
doses for a long-term study The sug cstion was also made that
one of the doses should approximate the occupational exposure
level if possible
Soecific Considerations when Anolvina Chemicals to the Skin
• I: -as agreed that use of the automatic pipette was an acceptaDle
and the rrost accurate method for delivery of the desired dose to
tre skin Spreading the test ma:erial ith the disposable pipet:c
tin ‘as recommended in those instances ‘here the material is
pattcuLarL viscous
• The consensus as that the “olume of application should be 50
for the mouse (not to exceed 100 4l) and a Limit of 300 pl for
tne rat
• The following points are to be con..idered tn seiecti a a “e tc e
Sotubilit’. and suspendability
- High volatility
- Toxicity
- Permeability
- Does not induce tumor formation
Ihicles discussed include acetone water, mineral oil, acetone
+ cvclohexare (I I). ethanol + water, and toluene If
occupational exposure to a chemical occurs in conju.iction with a
part:cular ehicle then that vehicle should be considered The
vehicle used in zne toxicity studies should oe the same as tha:
used in the absorption studies
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• The consensus was that the interscapular region is the desired
sije of apolication The area of application should not exceed
10% of the animal’s total body surface area
• Duration of Study , the consensus was that a dermal bioassay should
be a Lifetime study, that is, two years for mice rats or
hams t e r s
• Design of Study , the design of the study should attempt to
approximate continuous exposure The frequency of apolication
should be determined prior to or concurrent with the 90-day stud’;
Five applications/week is the preferred frequency of application
but 3 applications/week would be acceptable
The factors to be considered in determining dosing frequency
inc lude
- Irritation
- Effects of clipping
- Toxic ity
- Absorption, dist ibution and excretion of the test substance
ipecial Concerns that Arise when Using the Den iaL Route
Problems concerned with skin clipping/shaving
There was an agreement that nicking due to clipping should not be
a problem if you have proper technical expertise and that
irritation due to clipping is generally not a problem pro”ided
that a minimum of 24 hours is allowed for the skin to recover
before the next dosing of the animal It was agreed chit :he ha:r
c’.cle is not an issue for dermal bioassays
2 Hoc. to prevent exposure by routes other than dermal
The consensus was that some exposure by other routes is a oroblem
chat “we all have to live with” f we are going to study tne
to\citv/carcinogenicity of chemicals by the dermal route of
exposure Practical ways of minimizing exposure by other routes
are to house the animals individually (preferably in wire”nesh-
bottom cages) and to apply the chemical to the upper bact regior
Is c appropriate to minimize irritation/local toxiciti nV
alteration of the dosing scheduie ;nd site of applicatior
1€- the MTD of the test substance has been overestimated alter in
the concentration of the test material is preferable to nanging
the dosing schedule No one favored altering the site ot
application
When the test chemical is a liquid or is applied in solutton
should the dosage be expressed per surface area of per b dy
weight
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The consensus was that per body weight is the preferred way to
e’cpress dosage
5 When different dose levels of a chem i cal in solution are used, is
it preferab .e to keep the concentration the same and alter the
volume app lLed or to always apply the same volune and to adjust
the concentration
The consensus was that the volume should be kept constant and the
co-tcentration of the test solution should be altered when
different dose Levels are used
6 Should the Level, of exposure to the test material be constant as
the animal grows, and if so, how should this be done
Three options were proposed in answer to this question
a Keep a constant volume and alter the conce ition of the
dose solution as the body weight of the a 1. changes in
order to maintain a constant weight/body ..ght dosage
b Keep a constant concentration of dose solution and alter the
voluice as the body weight of the animal changes to maintain
a constant weight/body weight dosage
c Keep the dose solution volume and concentration constant
throughout the duratLon of testing
Tne consensus was that option “c” is realls the only practical.
optior but if the MTD is not based on irritation as the endpoint
then ootions “a” or “b” can be considered ‘nich option “c” dose
can st 1 11 be expressed as dose/body weigh: by using the total cose
adm i nistered/average bcdy weight throughout the study
There as also agreement on shich questions could not be ans ered at
‘ s time and on wnat follow up information is necessary in order tc arrLve at
: ese ans.ers
L A thorough literature search into tht use of rats and hamsters in
long’term carcinogenesis studies by the dermal. route, and the
results of these studies rr st be made Analysis of this database
snould then prov de the information needed for establishing the
usefulness of these animals as a second soecies for the dertral
bioas say
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2 A similar database on the relative susceptibility of different
mouse strains to skin irritation and to tumor induction following
application of chemicals to the skin is also needed !tice have
been used extensively in skin tuznorigenesis studies, so there
should be a wealth of data which, on analysis. would indicate
which mouse strains are the most appropriate for the purposes of
derrnal bioassay
3 It is imperacive that we establish a skin irritation scoring code
including hisropathologic evidence, for the rodent species to be
used in the long-term dermal bioassay This is especially needed
in the dose-selection phase The panel of experts were in
agreement that this scoring code should be arrived at in a second
workshop composed primarily of pathologists, but also including
toxicologists
4 The reasons for including toxicologists in this second workshop is
that it is anticipated that once scoring levels of irritation are
established this same group will also define that level of
irritation to be accepted as the endpoint in determing the MID
when skin is the target organ for toxicity
Recommendations for the Desizn of a Limited Dermal Protocol for the Screenina
of Acrylates/Methacrylates
The consensus was that a limited dermal bioassay was an acceptable
option for screening the carcinogenicit of acrylates/methacrvlaces it is to
be used to obtain a yes/no answer on the carcinogenicity of this class oi
members of this class, of chemicals and not for quantitative risk assessment
The acrvlates/methacrvlates have been shown to induce skin tumors and possibi
L-mphomas in mice, and therefore the need exists to look at both dermal ard
s-s:emic carcinogentc:t’. of these chemicals
• The consensus was to use one species, the mouse , for this iim::ed
protocol The strain of mouse will be selected after examtn ng
the dataoase in regards to sensitivit to skin irritation and
susceptinility to tumorigenesis of different mouse strains
• The duration of the study is not to be limited, that is it .s to
be at least an 18-month study
• It aas agreed to use only one sex , that is. r ale mice in this
limited protocol, since svszem c tumors have been found onL in
males, and no sex difference has been noted in skin tumor
suscept:bility with acrylates
• A 9O-da range.findtng study is recommended The number of doses
used depends on the chemical Preliminary acute studies are
imperatiie in order to pick the range of doses to be used in the
90-day range-finding study \‘ote Existing screening studies on
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acrylaces could be deemed adequate for screening purposes if ir
can be shown that an KTD had been achieved according co che
criteria to be established In che future EPA workshop |
In the 2 -year bioassay. che consensus was co use tvo doses
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