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) ------- 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. -2- ------- 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. -3- ------- 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. -4- ------- 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 .5- ------- 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. -6- ------- 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.). -7. ------- 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. -8- ------- 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. .9- ------- 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) 10- ------- 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 ------- 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 -12- ------- |