No. 441 

Technical Report Series 

NATIONAL TOXICOLOGY PROGRAM 

B6C3Fl MICE, SWISS (CD-l@) MICE, 

SKIN PAINT STUDIES OF 

COMPARATIVE INITIATION/PROMOTION 

AND SENCAR MICE 

National Institutes of Health 

Public Health Service 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 
FOREWORD 

applied research and to biological assay development and validation. 

relevant programs, staff, and resources from these Public Health Service agencies relating to basic and 

Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the 

Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control. In July 1981, the 

National Center for Toxicological Research (NCTR), Food and Drug Administration; and the National 

the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the 

Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; 

The National'Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of 

prevention of disease. 

chemicals. This knowledge is used for protecting the health of the American people and for the primary 

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous 

quality assurance audits before being presented for public review. 

Laboratory Practice Regulations, and all aspects of the chronic studies were subjected to retrospective 

and chronic studies were conducted in compliance with Food and Drug Administration (FDA) Good 

accordance with the Public Health Service Policy on Humane Care and Use of Animals. The prechronic 

exceed all applicable federal, state, and local health and safety regulations. Animal care and use were in 

were conducted in compliance with NTP laboratory health and safety requirements and must meet or 

The studies described in this Technical Report were performed under the direction of the NIEHS and 

studies. Selection per se is not an indicator of a chemical's carcinogenic potential. 

other species and quantitative risk analyses for humans require wider analyses beyond the purview of these 

this Technical Report are based only on the results of these NTP studies. Extrapolation of these results to 

human exposure, level of production, and chemical structure. The interpretive conclusions presented in 

Chemicals selected for NTP toxicology and carcinogenesis studies are chosen primarily on the bases of 

carcinogenic activity, of selected chemicals in laboratory animals (usually two species, rats and mice). 

These studies are designed and conducted to characterize and evaluate the toxicologic potential, including 

National Toxicology Program's World Wide Web site: http://ntp-server.niehs.nih.gov. 

Management. The Abstracts and other study information for 2-year studies are also available on the 

Listings of all published NTP reports and ongoing studies are also available from NTP Central Data 

Management, NIEHS, P.O. Box 12233, MD El-02, Research Triangle Park, NC 27709 (919-541-3419). 

copies of this Technical Report are available without charge while supplies last from NTP Central Data 

U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650). Single 

These NTP Technical Reports are available for sale from the National Technical Information Service, 
ON THE 

NTP TECHNICAL REPORT 

B6C3Fl MICE, SWISS (CD-l@) MICE, 

SKIN PAINT STUDIES OF 

COMPARATIVE INITIATION/PROMQTIQN 

AND SENCAR MICE 

Research Triangle Bark, NC 27709 

P.O. BOX 12233 

NATIONAL TOXICOLOGY PROGRAM 

February 1996 

NTP TR 441 

NIH Publication No. 96-3357 
2 Skin Paint Studies, NTP TR 441 

CONTRIBUTORS 

National Toxicology Program 

Evaluated and interpreted results and reported findings 

K.L. Witt, M.S., Oak Ridge Associated Universities 

D.B. Walters, Ph.D. 

G.S. Travlos, D.V.M. 

G.N. Rao, D.V.M., Ph.D. 

J.K. Haseman, Ph.D. 

T.J. Goehl, Ph.D. 

M.R. Elwell, D.V.M., Ph.D. 

W.C. Eastin, Ph.D. 

J.R. Bucher, Ph.D. 

D.A. Bridge, B.S. 

G.A. Boorman, D.V.M., Ph.D. 

C.J. Alden, Ph.D. 

Battelle Columbus Laboratories 

Conducted smdy, evaluated pathology findings 

R.L. Persing, D.V.M. 

J.D. Toft, 11, D.V.M., M.S. 

M.R. Hejtmancik, Ph.D., Study Director 

P.J. Kurtz, Ph.D., Principal Investigator ' 

Experimental Pathology Laboratories, Inc. 

K. Yoshitomi, D.V.M., Ph.D. 

J.F. Hardisty, D.V.M., Principal Investigator 

Provided pathology quality assurance 

Dynamac Corporation 

S. Brecher, Ph.D., Principal Investigator 

Prepared quality assurance audits 

NTP Pathology Working Group 

Evaluated slides, prepared pathology repon for B6C3FL and 

K. Yoshitomi, D.V.M., Ph.D. 

K. Takahashi, D.V.M., M.Sc., Ph.D. 

S.A. Stefanski, D.V.M. 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

J. Mahler, D.V.M. 

J.R. Hailey, D.V.M. 

J.C. Seely, D.V.M., Chair 

Swiss (CD-lm) mice (9 June 1992) 

Experimental Pathology Laboratories, Inc. 

National Toxicology Program 

National Toxicology Program 

National Toxicology Program 

National Toxicology Program 

National Toxicology Program 

PATHCO, Inc. 

Evaluated slides, prepared pathology report for B6C3Fl and 

K. Yoshitomi, D.V.M., Ph.D. 

R.C. Sills, D.V.M., Ph.D. 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

J.R. Hailey, D.V.M. 

J. Everitt, D.V.M. 

M.R. Elwell, D.V.M., Ph.D. 

J.C. Seely, D.V.M., Chair 

SENCAR mice (16 JUI) 1992) 

Experimental Pathology Laboratories, Inc. 

National Toxicology Program 

National Toxicology Program 

National Toxicology Program 

Chemical IndustIy Institute of Toxicology 

National Toxicology Program 

PATHCO, Inc. 
Skin Paint Studies, NTP TR 441 3 

SENCAR mice (1 3 August 1992) 

Evaluated slides, prepared pathology report for Swiss (CD-l@) and 

J.R. Hailey, D.V.M. 

PATHCO, Inc. 

J.C. Seely, D.V.M., Chair 

National Toxicology Program 

R.A. Herbert, D.V.M., Ph.D. 

National Toxicology Program 

Experimental Pathology Laboratories, Inc. 

KL Yoshitomi, D.V.M., Ph.D. 

Chemical Industry Institute of Toxicology 

D.L. Wolf, D.V.M., Ph.D. 

National Toxicology Program 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

National Toxicology Program 

J. Kanno, M.D., Ph.D. 

J.R. Hailey, D.V.M. 

PATHCO, Inc. 

J.C. Seely, D.V.M., Chair 

(1 October 1992) 

Evaluated slides, prepared pathology report for SENW mice 

National Toxicology Program 

R.A. Herbert, D.V.M., Ph.D. 

National Toxicology Program 

Experimental Pathology Laboratories, Inc. 

K. Yoshitomi, D.V.M., Ph.D. 

National Toxicology Program 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

North Carolina State University 

J. W. Ritchey, D.V.M., Observer 

National Toxicology Program 

J. Mahler, D.V.M. 

Analytical Sciences, Inc. 

Proviakd stathtical analyses 

S. Rosenblum, M.S. 

N.M. Garrison, B.S. 

R.W. Morris, M.S., Principal Investigator 

Biotechnical Services, Inc. 

Prepared Technical Report 

E.S. Rathman, M.S. 

S.R. Gunnels, M.A. 

J.R. Beverly, B.A. 

D.D. Lambright, Ph.D., Principal Investigator 
4 

CONTENTS 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE 

ABSTRACT .................................................................... 

7 

5 

.................................... 

............ 8 

INTRODUCTION ............................................................... 11 

MATERIALS AND METHODS ..................................................... 13 

RESULTS ..................................................................... 25 

DISCUSSION .................................................................. 97 

in the Comparative Initiation/Promotion Skin Paint Studies 

APPENDIX A Summary of Skin Lesions in Male and Female B6C3Fl Mice 

................... 101 

APPENDIX B Summary of Skin Lesions in Male and Female Swiss (CD-la) Mice 

in the Comparative InitiationPromotion Skin Paint Studies ................... 121 

APPENDIX C Summary of Skin Lesions in Male and Female SENCAR Mice 

in the Comparative Initiatioflromotion Skin Paint Studies ................... 141 

APPENDIX D Chemical Characterization and Dose Formulation Studies .................... 169 

APPENDIX E Ingredients. Nutrient Composition. and Contaminant Levels 

in NIH-07 Rat and Mouse Ration ....................................... 187 

APPENDIX F Sentinel Animal Program ............................................. 193 
5 

ABSTRACT 

genetic effects such as some promoting agents. 

short-term tests to detect agents that do not exert 

that here should be an increased emphasis on 

of chemical carcinogens. One recommendation was 

ogy Program (NTP) for the detection and evaluation 

methods that should be used by the National Toxicoltesting and evaluation recommended additional 

In 1983, an ad hoc panel on chemical carcinogenesis 

However, not all mouse strains are equally sensitive. 

than other commonly used laboratory rodent models. 

responsive (i.e., develops tumors using this protocol) 

opment. Mouse skin has been shown to be more 

motion) and the skin is monitored for tumor develtopical applications of one or more chemicals (proback of the skin (initiation) followed by repeated 

carcinogenic dose of a chemical is first applied to the 

study tumorigenesis. In one model, a topical subto identify chemicals with promoting potential and to 

Initiation/promotion models have been used routinely 

MNNG is not used as frequently for mouse skin 

(MNNG), a direct acting carcinogen, as the initiator. 

ducted using N-methyl-N’-nitro-N-nitrosoguanidine 

Juchau, 1980). Therefore, a second study was conmight not make this conversion (DiGiovanni and 

it was possible that the B6C3Fl mouse metabolism 

requires metabolic activation to achieve initiation and 

used to study tumorigenesis. However, DMBA 

promotion was selected because this pair is routinely 

and 12-0-tetradecanoylphorbol-13-acetate (TPA) 

7,12-dimethylbenz(a)anthracene (DMBA) initiation 

(CD-l@) and SENCAR mice. The combination of 

that of two often-used responsive strains, Swiss 

tumor response sensitivity of B6C3Fl mouse skin to 

initiators and promoters were used to compare the 

biology and response to chemical carcinogens, known 

carcinogenesis studies and much is known of its 

Since the B6C3Fl mouse is commonly used in NTP 

the initiation/promotion protocol was not known. 

The skin tumor response of the B6C3Fl mouse using 

cal as described on the following page. 

was used in combination with each promoting chemiused (Slaga et al., 1981). Each initiating chemical 

and known promoter after DMBA initiation, was also 

PA, benzoyl peroxide (BPO), a non-phorbol ester 

studies as is DMBA. In addition to the promoter 

tor controls) 

followed by repeated applications of acetone (initiaapplication of the initiator (DMBA or MNNG) 

treatment (promoter reference controls), or a single 

promoter (TPA or BPO) without prior initiation 

acetone (vehicle control), repeated applications of 

strain were treated with repeated applications of 

Additional groups of male and female mice of each 

least overall sensitivity to dermal carcinogenicity. 

mice was intermediate, and B6C3Fl mice showed the 

these criteria, the overall sensitivity of Swiss (CD-l@) 

SENCAR mice than in the other two strains. By 

develop earlier and with greater multiplicity in 

other two strains. Skin tumors also tended to 

required to produce effects equivalent to those in the 

lower doses of the test chemical were generally 

appeared to be SENCAR mice, in the sense that 

and BPO). The most sensitive of the three strains 

the chemicals under study (DMBA, MNNG, TPA, 

developing skin tumors after topical application of 

All three strains of mice demonstrated sensitivity by 

studies or effects on melanocytes are suspected). 

specific response observed in 2-year carcinogenicity 

tumor development (e.g. when there is a straintimes, be of more use in explaining mechanisms of 

protocol, promotion data from this strain may, at 

were less res.ponsive in the skin initiation/promotion 

to use for such studies. Though the B6C3Fl mice 

SENCAR mice would be the most acceptable strain 

sensitivity of various initiators and promoters, 

short-term tests and also on the skin tumor response 

In response to recommendations regarding specific 
Initiation/Promotiona Mouse Strain 

B6C3Fl Swiss (CD-le) SENCAR 

Design A 

50 pg DMBmA 

25 pg DMBA,TPA 

2.5 pg DMBmA 

0.25 pg DMBA,TPA~ 

2.5 pg DMBA/BPO~ 

25 pg DMBA/BPO 

1,000 pg MNNGmA 

100 pg MNNGmA 

Design B 

1,000 pg MNNGBPO 

500 pg MNNGBPO 

100 pg MNNGBPO 

100 pg MNNGf100 pg MNNG 

2.5 pg DMBAL2.5 pg DMBA 

Complete Carcinogend 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

x 

X 

a Mice received a single initiating application followed by repeated promotion applications for up to 52 weeks. 

Mice received repeated applications of DMBA and MNNG. 

BPO applications contained 20 mg BPO. 

B6C3Fl and Swiss (CD-le) mice received 5 pg TPA; SENCAR mice received 1 pg TPA 

more tumors per mouse than did Swiss (CD-l@) 

SENCAR mice developed skin tumors and averaged 

strains with both carcinogens. More B6C3Fl and 

was a high incidence of skin tumors in all three 

than in the initiation and promotion studies. There 

three strains under these conditions was more similar 

DMBA and MNNG. The skin tumor response in all 

applications of low concentrations of the carcinogens 

The 1-year complete carcinogen studies used repeated 

studies. 

(CD-l@) and SENCAR mice for complete carcinogen 

response of B6C3Fl mice was similar to Swiss 

compared to Swiss (CD-l@) and SENCAR mice, the 

sensitivity to the initiation/promotion protocol when 

Although B6C3Fl mice exhibited the lowest overall 

mice than in B6C3Fl and Swiss (CD-l@) mice. 

mice. Skin tumors developed earlier in SENCAR 

A summary of the Technical Reports Review Subcommittee comments and the public discussion on this Technical Report appears on 

Page 8- 
Skin Paint Studies, NTP TR 441 7 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE 

NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS 

responsibilities in reviewing NTP studies: 

representatives of any institution, company, or governmental agency. In this capacity, subcommittee members have four major 

promotion skin paint studies on June 21, 1994, are listed below. Subcommittee members serve as independent scientists, not as 

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on the comparative initiation/ 

l to ascertain that all relevant literature data have been adequately cited and interpreted, 

l to determine if the design and conditions of the NTF’ studies were appropriate, 

l to ensure that the Technical Report presents the experimental results and conclusions fully and clearly, and 

l to judge the significance of the experimental results by scientific criteria. 

Arnold L. Brown, M.D., Chair 

Madison, WI 

University of Wisconsin Medical School 

Paul T. Bailey, Ph.D., Principal Reviewer 

Princeton, NJ 

Stonybrook Laboratories, Inc. 

Meryl H. Karol, Ph.D. 

Pittsburgh, PA 

University of Pittsburgh 

Department of Environmental Occupational Health 

Curtis D. Klaassen, Ph.D. 

Kansas City, KS 

University of Kansas Medical Center 

Department of Pharmacology and Toxicology 

San Antonio, TX 

University of Texas Health Sciences Center 

Claudia S. Miller, M.D., Principal Reviewer 

Janardan K. Reddy, M.D. 

Chicago, IL 

Northwestern University Medical School 

Department of Pathology 

Irma RUSSO, M.D. 

Philadelphia, PA 

Fox Chase Cancer Center 

Boston, MA 

Dana-Farber Cancer Institute 

Harvard School of Public Health and 

Division of Biostatistics 

Louise Ryan, Ph.D., Principal Reviewer 

Robert E. Taylor, M.D., Ph.D. 

Washington, DC 

Howard University College of Medicine 

Department of Pharmacology 

Matthew J. van Zwieten, D.V.M., Ph.D. 

West Point, PA 

Merck Research Laboratories 

Department of Safety Assessment 

Mary Jo Vodicnik, Ph.D. 

Belgium 

Lilly MSG Development Center 

Jerrold M. Ward, D.V.M., Ph.D. 

Frederick, MD 

National Cancer Institute 
8 Skin Paint Studies, NTP TR 441 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS 

Park, NC. 

Environmental Health Sciences, Research Triangle 

review meeting was held at the National Institute of 

Technical Reports Review Subcommittee. The 

al Toxicology Program Board of Scientific Counselors 

SENCAR mice received public review by the Nationstudies of B6C3Fl mice, Swiss (CD-l@) mice, and 

these comparative initiation/promotion skin paint 

On June 21, 1994, the draft Technical Report on 

plete carcinogens. 

chemical initiators and promoters, as well as comSwiss (CD-l@) and SENCAR mice, using known 

skin to that of two often-used responsive strains, 

pare the tumor response sensitivity of B6C3Fl mouse 

the objectives of this research project were to comthe initiation protocol was not known. Therefore, 

large database exists, the skin tumor response using 

used for NTP carcinogenesis studies and for which a 

agents. For the B6C3F1 mouse, the strain commonly 

not exert genetic effects such as some promoting 

emphasis on short-term tests to detect agents that do 

by the NTP had recommended that the NTP increase 

carcinogenesis testing and evaluation commissioned 

relating that in 1983, an ad hoc panel on chemical 

Dr. W.C. Eastin, NIEHS, introduced the report by 

carcinogens. More B6C3Fl and SENCAR mice 

incidence of skin tumors in all three strains with both 

the initiation/promotion studies. There was a high 

response in all three strains was more similar than in 

the complete carcinogen studies, the skin tumor 

least overall sensitivity to dermal carcinogenicity. In 

mice was intermediate, and B6C3Fl mice showed the 

these criteria, the overall sensitivity of Swiss (CD-l@) 

SENCAR mice relative to the other two strains. By 

earlier and to exhibit increased multiplicity' in 

two strains. Skin tumors also tended to develop 

required to produce effects equivalent to the other 

sense that lower doses of test chemical were generally 

three strains appeared to be SENCAR mice, in the 

the concentrations tested, the most sensitive of the 

under study (DMBA, MNNG, TPA, and BPO). At 

tumors after topical applications of the chemicals 

of mice demonstrated sensitivity by developing skin 

For the initiation/promotion studies, all three strains 

and provided a detailed report of the study results. 

used for in-life data collection for these 1-year studies 

Dr. Eastin described the study design and techniques 

and Swiss (CD-l@) mice. 

developed earlier in SENCAR mice than in B6C3Fl 

mouse than did Swiss (CD-l@) mice. Skin tumors 

developed skin tumors and averaged more tumors per 

appearance of the first tumor, an in-life observation. 

responded that the analysis was based on the time of 

tumor was not done. Dr. J.K. Haseman, NIEHS, 

asked why a standard survival analysis on time to 

carcinogens (DMBA or MNNG). Dr. Ryan also 

promotion or those receiving repeated application of 

tumors only in groups receiving initiation with 

initiation. Dr. Eastin said there should have been 

the groups receiving TPA without DMBA or MNNG 

questioned the implication of tumors appearing in 

study after lesions had developed. Dr. Ryan also 

not really dying but were being removed from the 

Dr. Eastin explained that many of these animals were 

whether this complicated the statistical analyses. 

in the TPmA promoter reference group and 

sensitivity of the SENCAR strain in terms of survival 

should be some discussion regarding the increased 

Dr. Ryan, a principal reviewer, suggested that there 

reaction. 

the chemicals as evidenced by a marked inflammatory 

significantly more sensitive to the irritant effects of 

mice to tumor promotion were also those that were 

front of the report that the most sensitive strains of 

Dr. Bailey said there should be a statement in the 

advice on whether this model was a useful adjunct. 

responded that the NTP wanted the Subcommittee's 

the NTP toxicity/carcinogenesis studies. Dr. Eastin 

determine if this model would be a useful adjunct to 

provide mechanistic tumorigenesis data and to 

as stated in the report, these studies were designed to 

Dr. Bailey, the second principal reviewer, noted that, 

asked for a clearer explanation of why this study was 

other two strains should be discussed. Dr. Miller 

promoter in SENCAR mice as compared with the 

effect on the findings of the much lower dose of TPA 

affected the outcomes. Dr. Miller thought that the 

52 weeks, so he doubted that the error would have 

noted that the correct dose was given for 50 of the 

reference group upon the study results. Dr. Eastin 

effects of dose .errors in the DMBWA promoter 

there was a need for explanation of the possible 

Dr. Miller, the third principal reviewer, said that 
Skin Paint Studies, NTP TR 441 9 

performing such studies in B6C3Fl mice. 

conducted and what conclusions can be drawn about 

which was accepted unanimously with eleven votes. 

mice be accepted. Dr. Bailey seconded the motion, 

B6C3Fl mice, Swiss (CD-l@) mice, and SENCAR 

comparative initiation/promotion skin paint studies of 

Dr. Ryan moved that he Technical Report on 

Agency for Research on Cancer that dealt with 

and there was also a review by the International 

Dr. R. Griesemer, NIEHS, said that this was correct 

years ago by the Environmental Protection Agency. 

had been a forum or review of this subject several 

most test chemicals. Dr. Bailey thought that there 

should not be doing initiation/promotion studies on 

Dr. Reddy said that with limited resources, the NTP 

the NTP toxicology and carcinogenesis studies, 

model for providing mechanistic data as an adjunct to 

Regarding the usefulness of the initiation/promotion 

more predictive of what might happen in humans. 

mate goal being to develop information that will be 

target genes, or alternative methods, with the ultistudies of chemical interactions with receptors or 

initiation/promotion, transgenic mice, mechanistic 

select from a variety of possibilities, including 

NIEHS, stated that the NTP would like to be able to 

most promoters were organ specific. Dr. G. Lucier, 

predictive for humans. Dr. Reddy commented that 

test or species but rather the species or test most 

goal of toxicology was not to find the most sensitive 

mechanism. Dr. Klaassen cautioned that a major 

with thyroid tumors that act through a promotional 

rationale, for example, as with chemicals associated 

be more selective and based on some scientific 

Dr. Klaassen said use of this protocol would need to 

for standard initiation/promotion studies. 

ing cell cycle stage specificity might diminish priority 

not just skin. The newer approaches to understandinitiation/promotion in all organs where data existed, 
10 Skin Paint Studies, NTP TR 441 
11 

INTRODUCTION 

(NTP, 1984). 

detection and evaluation of chemical carcinogens 

recommend methods that NTP should use for the 

chemical carcinogenesis testing and evaluation to 

Scientific Counselors established an ad hac panel on 

The National Toxicology Program (NTP) Board of 

tinely used to study this process. 

Mouse skin initiation/promotion is one model rouducted to study the mechanism of tumorigenesis. 

cussed in their review was the research being conemphasized in these areas. One of the areas disas some promoting agents) and research should be 

detect agents that do not exert genetic effects (such 

there is a recognized deficiency of short-term tests to 

assays for genetic toxicity/potential carcinogenicity, 

and positive predictive value of the commonly used 

gress has been made in establishing the sensitivity 

The panel concluded that, while considerable proSubsequent treatment of initiated cells with a second, 

without further treatment does not produce tumors. 

treatment with a subthreshold dose of an initiator 

Quintanilla et al., 1986). In the mouse skin protocol, 

tumors after repeated exposure (Bizub el al., 1986; 

moter acts on these cells in some way to produce 

change to some epidermal cells and that the proresult of a mutagenic event that causes a heritable 

studies support the concept that initiation is the 

have been shown to exist (Slaga, 1983). More recent 

dence for multistep processes in other organ systems 

skin carcinogenesis is a multistep process and eviSlaga et aL, 1982). Now it is generally accepted that 

Hennings and Boutwell, 1969; VanDuuren, 1969; 

study the process of tumorigenesis (Boutwell, 1967; 

investigators have continued to use this protocol to 

used by Mottram (1944a,b). Since these early studies, 

croton oil was used to produce skin tumors, was first 

of a carcinogen followed by repeated applications of 

protocol, in which a single subthreshold application 

1941; Rous and Kidd, 1941). An initiation/promotion 

genesis (Berenblum, 1941; Berenblum and Shubik, 

were the first to suggest distinctive steps in tumoriHistorically, results of skin “painting” studies in mice 

et al., 1993; Warren et al., 1993). 

studies can be found in the literature (Hennings 

findings of the mouse skin initiafion/promotion 

ing these stages in tumorigenesis based on the 

frequency of genetic changes. Many reviews describa change believed to be associated with an increased 

but some of these can progress to a malignant tumor, 

papilloma formation. Papillomas are benign tumors, 

reversible and require continued exposure to cause 

initiation step, the effects of most promoters are 

benign squamous papillomas. In contrast to the 

neoplastic change, and result in the formation of 

can produce clonal expansion, expression of the 

noncarcinogenic agent having promotion potential 

carcinogenesis studies. 

model would be a useful adjunct to the NTP toxicity/ 

genesis data, it was of interest to determine if this 

studies are designed to provide mechanistic tumori- 

-promoting activities (Slaga, 1984). Because these 

ogen, i.e., whether it has both tumor-initiating and 

a compound but also whether it is a complete carcinmine the tumor-initiating and -promoting activities of 

This mouse skin model can be used not only to determouse strains (DiGiovanni, 1992). 

involve metabolic and genetic differences between 

initiation and promotion is not clear, but seems to 

The reason for differences in skin tumor response to 

untested strains using this protocol is not predictable. 

(Slaga and Fischer, 1983) and the tumor response of 

species, not all strains of mice are equally sensitive 

initiation/promotion studies than most other rodent 

While the mouse is a more responsive model for skin 

carcinogens. 

the different mouse strains to identify complete 

promotion studies and to compare the potential of 

(CD-l@) and SENCAR mice in topical initiation/ 

the tumor response of the B6C3Fl mouse with Swiss, 

studies. Thus these studies were designed to compare 

strain has not been used in initiation/promotion 

carcinogenesis studies using B6C3Fl mice, but this 

The NTP has directed more than 400 toxicity/ 
12 Skin Paint Studies, NTP TR 441 
13 

MATERIALS AND METHODS 

Sciences (NXEHS). 

at the National Institute of Environmental Health 

performed in support of the 1-year studies are on file 

firmed by the study laboratory. Reports on analyses 

Institute (Kansas City, MO) (Appendix D) and conanalytical chemistry laboratory, Midwest Research 

Identity and purity analyses were conducted by the 

America (Maple Shade, NJ) in one lot (WM-40). 

Benzoyl peroxide was obtained from Akzo Chemie 

Benzoyl Peroxide (CAS No. 94-36-0) 

CHARACTERIZATION 

PROCUREMENT AND 

approximately 99% when corrected for water content. 

area. The overall purity was determined to be 

impurities with areas totaling 0.4% of the major peak 

liquid chromatography revealed a major peak and two 

systems indicated one major spot. High-performance 

anhydrous basis. Thin-layer chromatography by two 

indicated a purity of 97% +- 2% calculated on an 

19.7% +- 0.5% water. Functional group titration 

Nuclear magnetic resonance spectroscopy indicated 

with the theoretical values for benzoyl peroxide. 

analyses for carbon and hydrogen were in agreement 

performance liquid chromatography. Elemental 

titration, thin-layer chromatography, and highmagnetic resonance spectroscopy, functional group 

was determined by elemental analyses, nuclear 

and nuclear magnetic resonance spectroscopy. Purity 

as benzoyl peroxide by infrared, ultravioletjvisible, 

The chemical, a white powdered solid, was identified 

chemical was observed. 

violet spectroscopy; no degradation of the bulk 

high-performance liquid chromatography and ultrachemical was monitored by the study laboratory using 

During the 1-year studies, the stability of the bulk 

Bulk benzoyl peroxide was stored under refrigeration. 

bulk chemical be stored protected from light at 5" C. 

analytical chemistry laboratory recommended that the 

Based on half-life data from the manufacturer the 

7,12-Dimethylbenz(a)anthracene 

NIEHS. 

support of the 1-year studies are on file at the 

(Appendix D). Reports on analyses performed in 

were conducted by the analytical chemistry laboratory 

laboratory. Identity, purity, and stability analyses 

(K-4) and was purified by the analytical chemistry 

Eastman Kodak Company (Rochester, NY) in one lot 

7,12-Dimethylbenz(a)anthracene was obtained from 

(CAS NO. 57-97-6) 

was determined to be greater than 99%. 

0.1% relative to the major peak. The overall purity 

major peak and no impurities with areas greater than 

chromatography using two systems indicated one 

spot and one trace impurity using two systems. Gas 

water. Thin-layer chromatography indicated a major 

Karl Fischer water analysis indicated less than 0.4% 

theoreticalvaluesfor7,12-dimethylbenz(a)anthracene. 

for carbon and hydrogen were in agreement with the 

raphy, and gas chromatography. Elemental analyses 

ses, Karl Fischer water analysis, thin-layer chromatogtroscopy. Purity was determined by elemental analyviolet/visible, and nuclear magnetic resonance spec7,12-dimethylbenz(a)anthracene by infrared, ultraThe chemical, a light yellow powder, was identified as 

observed. 

raphy. No degradation of the bulk chemical was 

using ultraviolet spectroscopy and gas chromatogcal was monitored periodically by the study laboratory 

amber glass bottles. The stability of the bulk chemibility, the bulk chemical was stored at -20" C in 

light at temperatures up to 60" C. To ensure stachemical for 2 weeks when stored protected from 

7,12-dimethylbenz(a)anthracene was stable as a bulk 

chromatography. These studies ndicated that 

by the analytical chemistry laboratory using gas 

Stability studies of the bulk chemical were performed 
14 Skin Paint Studies, NTP TR 441 

N-Methyl-N'-nitro-N-nitrosoguanidine 

NIEHS. 

support of the 1-year studies are on file at the 

(Appendix D). Reports on analyses performed in 

conducted by the analytical chemistry laboratory 

one lot (8228CK). Identity and purity analyses were 

from Aldrich Chemical Company (Milwaukee, WI) in 

N-Methyl-N'-nitro-N-nitrosoguanidine was obtained 

(CAS NO. 70-25-7) 

approximately 99%. 

peak area. The overall purity was determined to be 

impurities with areas greater than 0.1% of the major 

chromatography revealed a major peak and no 

ity by a second system. High-performance liquid 

major spot with a minor, a trace, and a slight impurand a minor spot by one system and indicated a 

Thin-layer chromatography indicated a major spot 

indicated that he water content was negligible. 

resonance spectroscopy and elemental analysis 

N'-nitro-N-nitrosoguanidine. Nuclear magnetic 

agreement with the theoretical values for N-methylanalyses for carbon, hydrogen, and nitrogen were in 

performance liquid chromatography. Elemental 

spectroscopy, thin-layer chromatography, and highelemental analyses, nuclear magnetic resonance 

resonance spectroscopy. Purity was determined by 

by infrared, ultraviolethrisible, and nuclear magnetic 

identified as N-methyl-N'-nitro-N-nitrosoguanidine 

The chemical, a light yellow crystalline solid, was 

NIEHS. 

support of the 1-year studies are on file at the 

(Appendix D). Reports on analyses performed in 

conducted by the analytical chemistry laboratory 

studies. Identity, purity, and stability analyses were 

lot (F-121). All five lots were used during the 1-year 

and F-121. 

and UN2811, and 99% for lots 00411999, 0E11999, 

overall purity was determined to be 97% for lots 031 

that were approximately 1% of the major peak. The 

in lots 00411999,0E11999, and F-121 with peak areas 

raphy indicated between two and five trace impurities 

the major peak. High-performance liquid chromatogrities with peak areas that were approximately 3% of 

UN2811 indicated between seven and 11 trace impuperformance liquid chromatography of lots 031 and 

one major peak in all five lots. In addition, highHigh-performance liquid chromatography indicated 

trace impurity, and two very slight trace impurities. 

second system identified a trace impurity, a slight 

impurity and a very slight race impurity, and a 

with one major spot, one system detected a trace 

one trace impurity was detected. For lot 031, along 

lots. For lot 00411999, along with one major spot, 

chromatography indicated one major spot for all five 

performance liquid chromatography. Thin-layer 

mined by thin-layer chromatography and highspectrometry. The purity of the five lots was deternuclear magnetic resonance spectroscopy and mass 

Each lot of the chemical was identified as TPA by 

dation of the bulk chemical was observed. 

12-0-Tetradecanoylphorbol-13-acetate 

L.C. Services Corporation (Woburn, MA) in one 

to assist in tracking. TPA was also obtained from 

chemical and was assigned a new number (UN2811) 

lot 00411999 was received from Pharmacia PL Bio- 

(00411999 and 0E11999). A second shipment of 

PL Biochemical (Milwaukee, WI) in two lots 

(Brewster, NY) in one lot (031) and from Pharmacia 

obtained from Consolidated Midland Corporation 

12-0-Tetradecanoylphorbol-13-acetate (TPA) was 

(CAS NO. 16561-29-8) 

was exposed to light and air. 

from light and for 3 hours when the dose formulation 

3 weeks at 5" C when stored in sealed vials protected 

the dose formulations was confirmed for at least 

performance liquid chromatography. The stability of 

the analytical chemistry laboratory using highstudies of the dose formulations were performed by 

benzoyl peroxide in acetone (Table Dl). Stability 

The dose formulations were prepared by dissolving 

Benzoyl Peroxide 

OF DOSE FORMULATIONS 

PREPARATION AND ANALYSIS 
Materials and Methods 15 

two of the four formulations (Table D3). 

with the results obtained by the study laboratory for 

the analytical chemistry laboratory were in agreement 

Results of the periodic referee analyses performed by 

(15/16) were within 10% of the target concentration. 

(Table D2). Of the dose formulations analyzed, 94% 

formulations were analyzed at least every 8 weeks 

performance liquid chromatography. The dose 

analytical chemistry laboratory using highperoxide were conducted at the study laboratory and 

Periodic analyses of the dose formulations of benzoyl 

and less than 3 hours when exposed to light and air. 

3 weeks when stored in the dark at room temperature 

the dose formulations was confirmed for at least 

performance liquid chromatography. The stability of 

analytical chemistry laboratory using high- 

(Table Dl). Stability studies were performed by the 

7,12-dimethylbenz(a)anthracene in acetone 

The dose formulations were prepared by dissolving 

7,12-Dimethylbenz(a)anthracene 

tory (Table D3). 

agreed with the results obtained by the study laboraperformed by the analytical chemistry laboratory 

concentration. Results of periodic referee analyses 

lations analyzed were within 10% of the target 

every 8 weeks (Table D2). All 23 of the dose formustudies, the dose formulations were analyzed at least 

tory using ultraviolet spectroscopy. During the 1-year 

the study laboratory and analytical chemistry labora7,12-dimethylbenz(a)anthracene were conducted at 

Periodic analyses of the dose formulations of 

to light and air. 

vials in the dark and for 3 hours when stored exposed 

at least 3 weeks stored at room temperature in sealed 

stability of the dose formulations was confirmed for 

using high-performance liquid chromatography. The 

were performed by the analytical chemistry laboratory 

(Table Dl). Stability studies of dose formulations 

N-methyl-N'-nitro-N-nitrosoguanidine in acetone 

The dose formulations were prepared by dissolving 

N-Methyl-N'-nitro-N-nitrosoguanidine 

matography. During the 1-year studies, the dose 

istry laboratory using high-performance liquid chroducted at the study laboratory and analytical chemN-methyl-N'-nitro-N-nitrosoguanidine were conPeriodic analyses of the dose formulations of 

obtained by the study laboratory (Table D3). 

analytical chemistry laboratory agreed with the results 

Results of periodic referee analyses performed by the 

(19/20) were within 10% of the target concentration. 

(Table D2). Of the dose formulations analyzed, 95% 

formulations were analyzed at least every 8 weeks 

when stored in amber glass bottles. 

confirmed for at least 3 weeks at room temperature 

raphy. The stability of the dose formulations was 

laboratory using high-performance liquid chromatogtions were performed by the analytical chemistry 

(Table Dl). Stability studies of the dose formula12-0-tetradecanoylphorbol-13-acetate in acetone 

The dose formulations were prepared by dissolving 

12-0-Tetradecanoylphorbol-13-acetate 

(Table D3). 

study laboratory for three of the six formulations 

laboratory agreed with the results obtained by the 

referee analyses performed by the analytical chemistry 

10% of the target concentration. Results of periodic 

dose formulations analyzed, 92% (24/26) were within 

analyzed at least every 8 weeks (Table D2). Of the 

During the 1-year studies, the dose formulations were 

tory using high-performance liquid chromatography. 

the study laboratory and analytical chemistry laboratetradecanoylphorbol-13-acetate were conducted at 

Periodic analyses of the dose formulations of 12-0- 

GENERAL STUDY DESIGN 

as the promoter treatment. Additional groups of 

weekly applications of either TPA, BPO, or acetone 

(MNNG) as the initiator treatment followed by 

tered N-methyl-N'-nitro-N-nitrosoguanidine 

groups of 30 male and 30 female mice were adminisfor the entire period of dosing. In study design B, 

weekly applications of acetone, TPA, BPO, or DMBA 

groups of 30 male and 30 female mice received 

or acetone as a promoter treatment. Additional 

phorbol-13-acetate (PA), benzoyl peroxide (BPO), 

weekly applications of either 12-0-tetradecanoyl- 

(DMBA) as an initiator treatment, followed by 

administered 7,12-dimethylbenz(a)anthracene 

design A, groups of 30 male and 30 female mice were 

known tumor initiators and promoters. In study 

mice, Swiss (CD-l@) mice, and SENCAR mice to 

conducted to compare the skin response of B6C3Fl 

Two 1-year studies (design A and design B) were 
16 Skin Paint Studies, NTP TR 441 

total dose of 20 mg per mouse. 

0.1 mL, each containing 10 mg BPO, to achieve the 

the application of two consecutive volumes of 

BPO in the vehicle, BPO doses were administered by 

volume of 0.1 mL. Due to relatively low solubility of 

TPA, and acetone doses were administered in a 

ning at week 1 of the study. All DMBA, MNNG, 

TPA, BPO, DMBA, or MNNG were treated begin51 weeks of dosing. Animals receiving only acetone, 

ment and weekly thereafter for the remaining 

dose was applied one week after the initiator treatduring the first week of dosing. The first promoter 

weekly thereafter. Initiators were applied once 

48 hours prior to the first chemical treatment and 

hair at the site of application was clipped 24 to 

applied to the intrascapular region of the back. The 

period of dosing. In both study designs doses were 

tions of acetone, PA, BPO, or MNNG for the entire 

30 male and 30 female mice received weekly applicatrations of 1 pg TPA. 

groups receiving TPA were restarted at dose concenwithout ulceration. Additional SENCAR mice 

dose per mouse gave a minimal hyperplastic response 

8 weeks. These studies indicated that a 1 pg TPA 

1, 2, or 5 pg TPA per mouse once a week for 

SENCAR mice received repeated applications of 0.5, 

in which groups of five male and five female 

evaluation. A dose range pilot study was performed 

groups were removed from the study without further 

irritation, scales, and ulcers. Animals from these 

repeated applications of 5 pg TPA had excessive 

skin application site of SENCAR mice receiving 

strains. However, after approximately 3 months, the 

prescribed TPA promoter doses of 5 pg for all mouse 

In both study designs A and B, the original protocol 

acetone topically once per week for 52 weeks. 

mice of each of the three strains were administered 

Vehicle Control: Groups of 30 male and 30 female 

the study. 

plied once per week for the remaining 51 weeks of 

followed by acetone as a promotion treatment apstrains), 25 pg (all strains), or 50 pg (B6C3Fl), 

of 0.25 pg [Swiss (CD-l@) and SENCAR], 2.5 pg (all 

mice were administered single DMBA initiation doses 

Initiator Reference: Groups of 30 male and 30 female 

promotion treatment in week 2. 

B6C3Fl mice received one dose of 16 mg BPO as a 

all mice were initiated with 16 mg BPO, and all 

Due to a dose formulation error in study design A, 

(CD-l@)], or 20 mg BPO (all strains) for 52 weeks. 

TPA (SENCAR), 5 pg TPA [B6C3Fl and Swiss 

30 female mice received topical applications of 1 pg 

Promoter Reference: Groups of 30 male and 

of the study. 

treatment once per week for the remaining 51 weeks 

(CD-l@)] or 1 pg TPA (SENCAR) as a promotion 

administration of 5 pg TPA [B6C3Fl and Swiss 

50 pg (B6C3FJ. Initiation was followed by the 

SENCAR], 2.5 pg (all strains), 25 pg (all strains), or 

initiation doses of 0.25 pg [Swiss (CD-l@) and 

30 female mice were administered single DMBA 

InitiationlPrornotion: Groups of 30 male and 

once per week for the remaining 51 weeks. 

in the first week of the study followed by 20 mg BPO 

strains) were administered 2.5 or 25 pg DMBA once 

Additional groups of 30 male and 30 female mice (all 

conceptual groups (Table 1): 

treatments in study design A can be ordered into five 2.5 pg DMBA topically as a complete carcinogen 

Thevarious combinations of initiation and promotion 30 female mice of all three strains were administered 

Study Design A Complete Carcinogen: Groups of 30 male and 

once per week for 52 weeks. 
Materials and Methods 17 

Design A in the Comparative Initiatioflromotion Skin Paint Studies: DMBA Initiationa 

TABLE 1 

Mouse Strain 

B6C3Fl Swiss (CD-l@) SENCAR 

Initiatiw/Promotion Treatmentb>' 

Vehicle Control 

AcetonelAcetone 

Initiator Reference 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

Promoter Reference 

20 mg BPOnO mg BPO 

5 pg TPN5 pg TPA 

1 pg TPAn fig TPAd 

Initiation/Promotion 

25 pg DMBN1 pg TPA 

2.5 pg DMBN1 pg PA 

0.25 pg DMBAn Kg TPA 

50 pg DMBN5 pg TPA 

25 pg DMBN5 pg TPA 

2.5 pg DMBN5 pg PA 

0.25 pg DMBN5 pg TPA 

25 pg DMBAnO mg BPO 

2.5 pg DMBAnO mg BPO 

Complete Carcinogen 

2.5 pg DMBAD.5 pg DMBA 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

All dose volumes were 0.1 mL except for BPO doses which were delivered as two 0.1 mL volumes containing half the total dose. 

a 30 males and 30 females/group 

administered 5 pg "'PA were replaced with groups receiving 1 pg TPA 

After 3 months of dosing, SENCAR mice were determined to be intolerant of the 5 pg TPA dose. Thus, SENCAR groups 

once weekly for the remaining 51 weeks of dosing. 

' Initiators were applied once during the first week of dosing; promoters were applied one week after initiator application, and then 
18 Skin Paint Studies, NTP TR 441 

the study. 

applied once per week for the remaining 51 weeks of 

followed by acetone as a promotion treatment 

1,000 pg MNNG in a single dose during week 1, 

mice (all strains) were administered 100, 500, or 

Initiator Reference: Groups of 30 male and 30 female 

51 weeks. 

(SENCAR) once per week for the remaining 

[B6C3Fl and Swiss (CD-l@)] or .1 pg TPA 

the first week of the study followed by 5 pg TPA 

were administered 100 or 1,000 pg MNNG once in 

Additional groups of 30 male and 30 female mice 

Promoter Reference: Groups of 30 male and Complete Carcinogen: Groups of 30 male and 

30 female mice received topical application of 1 pg 30 female mice of all three strains were administered 

TPA (SENCAR), 5 pg TPA [B6C3Fl and Swiss 100 pug MNNG topically as a complete carcinogen 

(CD-la)], or 20 mg BPO (all strains) for 52 weeks. once per week for 52 weeks. 
Materials and Methods 19 

Design B in the Comparative Initiation/Promotion Skin Paint Studies: MNNG Initiationa 

TABLE 2 

Mouse Strain 

B6C3Fl swiss (CD-la) SENCAR 

InitiatiordPromotion Treatmentbyc 

Vehicle Control 

Acetone/Acetone 

Initiator Reference 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

Promoter Reference 

20 mg BPOEO mg BPO 

5 pg TPN5 fig TPA 

1 pg TPN1 kg PAd 

InitiationlPromotion 

1,000 pg MNNGEO mg BPO 

500 pg MNNGEO mg BPO 

100 pg MNNGDO mg BPO 

1,000 pg MNNG/5 pg TPA 

100 pg MNNG/5 pg TPA 

1,000 pg MNNG/1 pg 'PA 

100 pg MNNG/l kg "A 

Complete Carcinogen 

100 pg MNNGfl00 pg MNNG 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

a 30 males and 30 females/group 

administered 5 pg TPA were replaced with groups receiving 1 pg TPA 

After 3 months of dosing, SENCAR mice were determined to be intolerant of the 5 pg TPA dose. Thus, SENCAR groups 

once weekly for the remaining 51 weeks of dosing. 

Initiators were applied once during the first week of dosing; promoters were applied one week after initiator application, and then 

All dose volumes were 0.1 mL except for BPO doses which were delivered as two 0.1 mL volumes containing half the total dose. 
20 Skin Paint Studies, NTP TR 441 

(Appendix F). 

cols of the NTP Sentinel Animal Program 

monitored during the studies according to the protoning of the studies. The health of the animals was 

Mice were approximately 8 weeks old at the beginSerology samples were collected for viral screening. 

parasite evaluation and gross observation of disease. 

mice per strain per study design were selected for 

beginning of the studies. Five male and five female 

Mice were quarantined for 27 to 30 days before the 

Laboratories (Portage, MI) for use in the studies. 

mice were obtained from Charles River Breeding 

(Frederick, MD) and male and female Swiss (CD-l@) 

obtained from Frederick Cancer Research Facility 

Male and female B6C3Fl and SENCAR mice were 

Source and Specification of Animals 

and contaminants is provided in Appendix E. 

given in Table 3. Information on feed composition 

biweekly. Further details of animal maintenance are 

available ad libitum. Cages and racks were rotated 

Mice were housed individually. Feed and water were 

Animal Maintenance 

monthly thereafter. 

weekly during the first three months of the study and 

findings were recorded weekly; animals were weighed 

All animals were observed twice daily. Clinical 

Clinical Examinations and Pathology 

System (TDMS). 

to NIEHS using the Toxicology Data Management 

were directly entered onto diskettes and transmitted 

tissue was called a carcinoma. Clinical observations 

necrotic in appearance and attached to underlying 

was called a papilloma. A papilloma that became 

least 2 mm in diameter and was present for 14 days 

recorded as a tissue mass. A tissue mass that was at 

Thus, at first appearance, each skin tumor was 

how the skin tumor response would be recorded. 

observations, guidelines were developed to define 

development. To provide some consistency for these 

follow the appearance and progression of any tumor 

Clinical observations of the skin were recorded to 

before affecting internal organs. 

would be expected to manifest itself at this site 

primary target organ, the effect of chemical treatment 

ment can be observed. Since the skin was the 

the appearance and progression of tumor developUsing the mouse skin initiation/promotion protocol, 

tory animal veterinarian, were killed. 

indicating pain or suffering, as judged by the laborawith ulcerations, debilitating conditions, or conditions 

interfered with feed or water consumption, and mice 

Mice with large masses or other conditions that 

aggressive moribund sacrifice policy was maintained. 

dosed mice would develop lesions on the skin and an 

In the present studies, it was expected that some 

of application and untreated) (Table 3). 

pathologic examinations were performed on skin (site 

preserved in 10% neutral buffered formalin. Histovisible lesions, and all major tissues were fixed and 

all organs and tissues were examined for grossly 

A necropsy was performed on all mice. At necropsy, 

untreated sites. 

the site of application as well as samples from 

assessment pathologist reviewed samples of skin from 

and the histotechnique was evaluated. A quality 

for accuracy, the slide and tissue counts were verified, 

individual animal records and tables were compared 

independent quality assessment laboratory. The 

records, and pathology tables were evaluated by an 

tissue audit. The slides, individual animal data 

Archives for inventory, slideblock match, and wet 

blocks, and residual wet tissues were sent to the NTP 

entered into TDMS. The microscopic slides, paraffin 

laboratory pathologist, and the pathology data were 

Microscopic evaluations were completed by the study 

dures have been described, in part, by Maronpot and 

ogists and the PWG. Details of these review procediagnoses represent a consensus of contractor patholgist, the diagnosis was changed. Thus, the final 

differed from the opinion of the laboratory patholorendered diagnoses. When the PWG consensus 

without any knowledge of dose groups or previously 

logic pathology. This group examined the tissues 

and other pathologists experienced in rodent toxicoPWG consisted of the quality assessment pathologist 

presented by the chair to the PWG for review. The 

pathologist, or lesions of general interest were 

ses between the laboratory and quality assessment 

administration, examples of disagreements in diagnoing examples of lesions related to chemical 

existed. Representative histopathology slides containthe laboratory and quality assessment pathologists 

tissues for which adisagreement in diagnosis between 

chair, who reviewed the selected tissues and any other 

ted to the NTP Pathology Working Group (PWG) 

The quality assessment report and slides were submit- 
Materials and Methods 21 

McConnell et al. (1986). 

separately or combined according to the guidelines of 

diagnosed lesions for each tissue type were evaluated 

subsequent analyses of the pathology data, the, 

Boorman (1982) and Boorman et al. (1985). For 

analyses are two sided. 

trends. All reported P values for the survival 

Tarone’s (1975) life table test to identify dose-related 

method for testing two groups for equality and 

dose-related effects on survival used Cox’s (1972) 

were not censored. Statistical analyses for possible 

survival analyses; animals dying from natural causes 

those missing or missexed were censored from the 

Animals found dead of other than natural causes and 

product-limit procedure of Kaplan and Meier (1958). 

The probability of survival was estimated by the 

Survival Analyses 

Statistical Methods 

performed. 

number of animals on which a necropsy was 

sites of occurrence, the denominators consist of the 

evaluation, or when neoplasms had multiple potential 

as is the case for skin tissue, before microscopic 

scopic examination was required to detect neoplasms, 

examined microscopically. However, when macrogiven as the numbers of animals affected at each site 

most neoplasms and all nonneoplastic lesions are 

calculation of statistical significance, the incidences of 

animals with that site examined microscopically. For 

lesions at a specific anatomic site and the number of 

are given as the number of animals bearing such 

as presented in Tables Al, A3, B1, B3, C1, and C3 

The incidences of neoplasms or nonneoplastic lesions 

Calculation of Incidence 

1972; Tarone, 1975) based on the observed time of 

tumors observed in-life was a life table test (Cox, 

quently, the primary statistical analysis for skin 

time of skin tumor onset for each animal. Consetumors, it was possible to determine the approximate 

once a week and included the presence of skin 

Because clinical observations were rcptinely recorded 

Analysis of Tumor Incidences 

were also present at the time of death of the animal. 

tumor onset. Most (but not all) of these tumors 

to Haseman (1984). 

further discussion of these statistical methods, refer 

to evaluate selected nonneoplastic lesions. For 

described in the preceding paragraphs were also used 

reported P values are one sided. The procedures 

were used in the analysis of tumor incidence, and 

overall dose-related trend. Continuity-corrected tests 

each exposed group with controls and a test for an 

Tests of significance included pairwise comp’arisons of 

as a logistic function of chemical exposure and time. 

which nonneoplastic lesion prevalence was modeled 

analysis used was a logistic regression analysis in 

death or not rapidly lethal, the primary statistical 

were considered to be incidental to the cause of 

Because all nonneoplastic lesions in these studies 

Analysis of Nonneoplastic Lesion Incidences 

Wolfe, 1973). 

cance using the Mann-Whitney U test (Hollander and 

The number of papillomas was analyzed for signifimultiple comparison procedures of Dunnett (1955). 

distributions, were analyzed using the parametric 

Body weight data, which have approximately normal 

Analysis of Continuous Variables 

this Technical Report. 

were otherwise addressed during the preparation of 

NTP staff, so all comments had been resolved or 

The audit findings were reviewed and assessed by 

presented in the reports and are on file at NIEHS. 

conducted. Audit procedures and findings are 

tables, and a draft of this NTP Technical Report were 

pathology data, pathology specimens, final pathology 

audits covering completeness and accuracy of the 

independent quality assurance contractor. Separate 

Archives, the study was audited retrospectively by an 

records from the study were submitted to the NTP 

Regulations (21 CFR, Part 58). In addition, as 

and Drug Administration Good Laboratory Practice 

The study was conducted in compliance with Food 

Quality Assurance Methods 
22 Skin Paint Studies, NTP TR 441 

Skin Paint Studies of B6C3Fl Mice, Swiss (CD-la) Mice, and SENCAR Mice 

Experimental Design and Materials and Methods in the Comparative Initiation/Promotion 

TABLE 3 

B6C3Fl Swiss (CD-1") SENCAR 

Battelle Columbus (Columbus, OH) 

Study Laboratory 

(Frederick, MD) 

Frederick Cancer Research Facility 

Animal Source 

30 males and 30 females 

Size of Study Groups 

B: 29 days 

A: 27 days 

Time Held Before Studies 

B: 57 days 

A 56 days 

Average Age When Studies Began 

B: 22 May 1985 

A: 22 April 1985 

Date of First Dose 

A: Initiator dose lx, 22 April 1985; 

Duration of Dosing 

14 April 1986 

51 weeks, 29 April 1985 - 

Promoter doses, lx weekly for 

14 May 1986 

51 weeks, 29 May 1985 - Promoter doses lx weekly for 

B: Initiator dose lx, 22 May 1985; 

B 14 May 1986 

A: 14 April 1986 

Date of Last Dose 

Battelle Columbus (Columbus, OH) 

(Portage, MI) 

Charles River Breeding Laboratories 

30 males and 30 females 

A: 29 days 

E: 29 days 

A: 58days 

B: 58 days 

A: 3 May 1985 

B: 31 May 1985 

25 April 1986 

51 weeks, 10 May 1985 - 

Promoter doses, lx weekly for 

A: Initiator dose lx, 3 May 1985; 

23 May 1986 

51 weeks, 7 June 1985 - 

Promoter doses lx weekly for 

B: Initiator dose lx, 31 May 1985; 

E: 23 May 1986 

A: 25 April 1986 

Battelle Columbus (Columbus, OH) 

(Frederick, MD) 

Frederick Cancer Research Facility 

30 males and 30 females 

Restart: 29 days 

B: 30 days 

A 30 days 

Restart: 57 days 

B: 58 days 

A: 58days 

Restart: 16 October 1985 

B: 6June1985 

A: 9 May 1985 

1 May 1986 

51 weeks, 16 May 1985 - Promoter doses, lx weekly for 

A Initiator dose lx, 9 May 1985; 

29 May 1986 

51 weeks, 13 June 1985 - 

Promoter doses lx weekly for 

B: Initiator dose lx, 6 June 1985; 

lx weekly for 51 weeks, 

16 October 1985; Promoter doses 

Restart: Initiator dose lx, 

1986 

h. 23 October 1985 - 8 October 

< 

c 

Restart: 8 October 1986 

B: 29 May 1986 

A: 1 May 1986 
Materials and Methods 23 

Skin Paint Studies of B6C3Fl Mice, Swiss (CD-la) Mice, and SENCAR Mice (continued) 

Experimental Design and Materials and Methods in the Comparative Initiatioflromotion 

TABLE 3 

B6C3F, Swiss (CD-la) SENCAR 

Carbon dioxide asphyxiation 

Method of Sacrifice 

B: 19-23 May 1986 

Necropsy Dates 

A: 21-25 April 1986 

60-61 weeks 

Average Age at Necropsy 

generated randomization procedure 

mean body weight by a computer 

groups of approximately equal initial 

Animals were randomly assigned to 

Method of Animal Distribution 

1 

Animals per Cage 

Toe mark 

Method of Identification 

ad libiturn; changed weekly 

Brothers, Gardners, PA), available 

NIH-07 open formula meal (Zeigler 

Diet 

120 days 

Maximum Storage Time for Feed 

Products, Inc., Garfield, NJ) 

Stainless-steel hopper-type (Lab 

Feeders 

system; available ad libitum 

supply) via automatic watering 

Tap water (Columbus municipal 

Water 

Garfield, NJ); changed weekly 

Polycarbonate (Lab Products Inc., 

Cages 

Corp., Warrensburg, NY) 

BetaChipse (Northeastern Products 

Bedding 

Carbon dioxide asphyxiation 

A: 28 April - 2 May 1986 

B: 27-30 May 1986 

60-61 weeks 

Same as B6C3Fl 

1 

Toe mark 

Same as B6C3F, 

120 days 

Same as B6C3Fl 

Same as B6C3Fl 

Same as B6C3Fl 

Same as B6C3Fl 

Carbon dioxide asphyxiation 

Restart: 13 October 1986 

B: 2-4 June 1986 

A: 5-8 May 1986 

60-61 weeks 

Same as B6C3Fl 

1 

Toe mark 

Same as B6C3Fl 

120 days 

Same as B6C3Fl 

Same as B6C3Fl 

Same as B6C3Fl 

Same as B6C3Fl 
24 Skin Paint Studies, NTP TR 441 

OH); changed evey 2 weeks 

(Snow Filtration Co., Cincinnati, 

Spun-bonded DuPont 2024 polyester Same as B6C3F1 

Cage Filters 

mely 2 weeks 

Products Inc., Garfield, NJ); changed 

Stainless Steel drawer-type (Lab 

RaCb 

Same as B6C3F1 

Same as B6C3F1 

Same as B6C3Fl 

Animal Room Environment 

Room air changes: 15 changeshour Room air changes: 15 changeshour Room air changes: 15 changeshour 

Fluorescent light: 12 hours/day Fluorescent light: 12 hours/day Fluorescent light: 12 hours/day 

Relative humidiry: 35%-65% Relative humidity: 35%-65% Relative humidity: 35%-65% 

Temperature: 21"-24' C Temperature: 21"-24" C Temperature: 21"-24" C 

See Tables 1 and 2 

Doses 

See Tables 1 and 2 See Tables 1 and 2 

clinical findings recorded weekly clinical findings recorded weekly clinical findings recorded weekly 

three months, and monthly thereafter; three months, and monthly thereafter; three months, and monthly thereafter; 

were weighed weekly during the first were weighed weekly during the first were weighed weekly during the first 

Animals observed twice daily; animals Animals observed twice daily; animals Animals observed twice daily; animals 

Type and Frequency of Observation 

Necropsy performed on all animals 

Necropsy 

from untreated sites. from ted 

skin from the site of application and skin from the site of application and skin from the site of application and 

Histopathology was performed on Histopathology was performed on Histopathology was performed on 

Histopathology 

Necropsy performed on all animals Necropsy performed on all animals 
25 

RESULTS 

PROTOCOL CONTROL GROUPS 

STUDY DESIGN A 

without DMBA initiation (Table 1). 

1 pglmouse in SENCAR] or BPO (20 mglmouse) 

[5 pglmouse in B6C3Fl and Swiss (CD-l@) and 

repeated topical applications of either TPA 

non-promoter acetone, and groups that received 

B6C3Fl] followed by repeated applications of the 

and SENCAR and 2.5, 25, or 50 pglmouse in 

DMBA [0.25, 2.5, or 25 pglmouse in Swiss (CD-l@) 

a single application of one of three concentrations of 

acetone vehicle alone, as well as groups that received 

male and female mice of each strain that received the 

functioned. These control groups included groups of 

how well this mouse skin initiation/promotion model 

were included in the study design as a measure of 

number of initiator and promoter control groups 

benzoyl peroxide (BPO) were used as promoters. A 

12-0-tetradecanoylphorbol-13-acetate (TPA) and 

anthracene (DMBA) was used as the initiator, and 

tumors. In study design A, 7,12-dimethylbenz(a)- 

a promoter without initiation should not produce 

a carcinogen without promotion, or repeated doses of 

vehicle alone, a single initiating subthreshold dose of 

assumptions of the model, topical applications of 

potential will produce skin tumors. Based on the 

repeated doses of a second chemical with promotion 

topical application of a carcinogen followed by 

this model, the combination of a single subthreshold 

chemicals with skin tumor promotion potential. In 

The mouse skin initiation/promotion model identifies 

vehicle controls of the respective strain (Table 4). 

tions followed by acetone was similar to that of the 

receiving any of the three initiator DMBA concentraFor each strain, survival of male and female mice 

Survival of Protocol Controls 

the vehicle controls (Table 4). 

initiation, survival was significantly lower than that of 

receiving repeated applications of TPA without prior 

However, in male and female SENCAR groups 

to that of the vehicle controls of the same strain. 

of TPA without prior initiation, survival was similar 

Swiss (CD-l@) groups receiving repeated applications 

control groups. In male and female B6OF1 and 

was also similar to that of the respective vehicle 

repeated applications of BPO without prior initiation 

Survival of males and females of each strain receiving 

controls (Table 4). 

groups were similar to those of the respective vehicle 

Final mean body weights of the protocol control 

Protocol Controls 

Body Weights and Clinical Findings in 

ulcer. 

irritation and no more than 12% developed signs of 

7% of male Swiss (CD-l@) mice developed signs of 

receiving repeated applications of BPO. Less than 

SENCAR mice or female Swiss (CD-l@) mice 

the site of application in male or female B6C3Fl or 

after (Table 5). Clinical signs were not observed at 

weeks 24 and 28, and decreased in incidence thereappeared after about 10 to 12 weeks, peaked between 

tion throughout the study and with ulcers that 

applications of TPA were observed with skin irrita- 

(CD-l@) and SENCAR mice receiving repeated 

controls. Some non-initiated male and female Swiss 

three strains were similar to those in the vehicle 

male and female DMBA initiator control mice of all 

The incidences of these clinical signs of toxicity in 

localized skin surface excavation and/or broken skin. 

inflamed or appearing sore. Ulcer was defined as 

ulcers. Irritation was defined as the skin being 

The primary clinical findings included irritation and 
26 Skin Paint Studies, NTP TR 441 

in the Comparative Initiatioflromotion Skin Paint Studies: Protocol Controls for Study Design A 

Survival and Body Weights of B6C3Fl Mice, Swiss (CD-1") Mice, and SENCAR Mice 

TABLE 4 

to Controls (%) 

Relative 

Final Weight 

Initiator/Promoter Survivala Final Mean Body Weightb (g) 

20 mg BPODO rng BPO 

5 pg TPN5 pg "PA 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

AcetonelAcetone 

Male 

B6C3Fl 

30130 

29/30 

30/30 

29/30 

30/30 

26/30 

20 mg BPO/20 mg BPO 

5 pg TPN5 pg TPA 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

AcetonelAcetone 

Female 

27/30 

30/30 

30/30 

30/30 

29/30 

30/30 

20 rng BPOnO mg BPO 

5 pg TPN5 pg TPA 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

AcetonelAcetone 

Male 

Swiss (CD-lm) 

20 mg BPO/2O mg BPO 

5 pg TPN5 pg TPA 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

AcetonelAcetone 

Female 

25/30 

21/30 

23/30 

29/30 

25/30 

27/30 

3OBO 

23/30 

28/30 

29/30 

29/30 

27/30 

48.4 f 0.6 

46.8 2 0.8 

48.3 2 0.6 

47.6 f 0.5 

47.0 f 0.6 

46.2 f 0.8 

43.1 2 0.9 

42.0 f 0.9 

46.5 f 0.9 

45.8 f 1.0 

45.5 f 1.1 

44.4 f 0.9 

51.6 f 1.3 

47.5 2 1.4 

51.4 f 1.2 

51.7 f 1.1 

51.7 f 1.2 

50.0 f 1.3 

40.8 & 1.1 

38.2 f 0.8 

41.0 2 1.3 

39.3 f 1.1 

40.4 f 1.2 

40.1 k 1.5 

- 

95 

97 

97 

100 

98 

97 

94 

105 

103 

102 

103 

95 

103 

103 

103 

102 

95 

102 

98 

101 

(continued) 
Results 27 

(continued) 

in the Comparative Initiatioflromotion Skin Paint Studies: Protocol Controls for Study Design A 

Survival and Body Weights of B6C3Fl Mice, Swiss (CD-ls) Mice, and SENCAR Mice 

TABLE 4 

to Controls (%) 

Relative 

Final Weight 

Initiator/Promoter Survival Final Mean Body Weight (9) 

20 mg BPOm mg BPO 

1 fig TPN1 fig TPA 

25 pg DMBAfAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

AcetonefAcetone' 

AcetonefAcetone 

Male 

SENCAR 

20 mg BPO/2O mg BPO 

1 kg "An fig 'PA 

25 kg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

htonefketone' 

AcetonefAcetone 

Female 

30/31 

27/29 

27/30 

29/30 

28/30 

23/30 

9/30; * 

28/30 

10/30** 

28/29 

3OL31 

29/30 

27/30 

27/30 

50.7 & 1.4 

48.8 f 2.8 

52.1 & 1.4 

54.1 ? 1.2 

54.0 ? 1.6 

51.7 f 1.4 

51.7 & 1.3 

41.1 f 1.2 

44.1 f 2.6 

44.7 f 1.4 

43.8 f 1.4 

43.0 f 1.4 

45.7 f 1.7 

44.5 f 1.4 

98 

94 

101 

105 

104 

92 

97 

100 

99 

97 

Vehicle control group for TPA restart 

Weights are given as mean k standard error. Differences from the vehicle control group are not significant by Dunnett's test. 

a Number of animals suwivinghumber initialIy in group. Subsequent calculations are based on animals surviving to the end of the 

** Significantly different (PSO.01) from the vehicle control group by life table pairwise comparison 

study. 
28 Skin Paint Studies, NTP TR 441 

Protocol Controls for Study Design A 

and SENCAR Mice in the Comparative InitiatiodPromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3Fl Mice, Swiss (CD-1") Mice, 

TABLE 5 

irritation Ulcer 

Week of Study 1-10 11-20 21-30 31-40 41-50 1-10 11-20 21-30 31-40 41-50 

20 mg BPOnO mg BPO 

5 pg TPN5 pg TPA 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

AcetonelAcetone 

B6C3Fl 

Male 

20 mg BPODO mg BPO 

5 pg TPN5 pg TPA 

50 bg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

AcetonelAcetone 

Female 

20 mg BPOm mg BPO 

5 pg TPN5 pg TPA 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

AcetonelAcetone 

Male 

Swiss (CD-lm) 

20 mg BPODO mg BPO 

5 pg TPN5 pg TPA 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

AcetonelAcetone 

Female 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 3 3 0 0 

2 3 3 5 4 

0 0 0 0 0 

2 2 0 0 0 

0 0 1 3 3 

1 6 9 13 19 

0 0 1 0 6 

0 0 0 0 1 

0 0 0 0 0 

1 2 0 0 0 

0 0 0 0 0 

0 2 5 19 23 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 1 0 1 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 1 0 0 3 

0 0 0 1 0 

2 3 3 1 0 

2 2 0 0 0 

1 0 1 4 3 

1 2 3 0 0 

2 1 4 13 9 

28 33 40 40 22 

2 0 0 0 1 

2819242523 

0 0 0 0 1 

3 1 0 1 1 

0 0 0 0 0 

1 0 1 2 0 
Results 29 

Protocol Controls for Study Design A (continued) 

and SENCAR Mice in the Comparative InitiationPromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3F, Mice, Swiss (CD-l@) Mice, 

TABLE 5 

Irritation Ulcer 

Week of Study 1-10 11-20 21-30 31-40 41-SO 1-10 11-20 21-30 31-40 41-50 

20 mg BPOEO mg BPO 

1 pg TPN1 pg TPA 

25 pg DMBNAcetone 

2.5 pg DMBAlAcetone 

0.25 pg DMBNAcetone 

AcetonelAcetone 

AcetonelAcetone 

Male 

SENCAR 

20 mg BPOnO mg BPO 

1 cg "TAD fig PA 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 Kg DMBNAcetone 

AcetonelAcetone 

AcetonelAcetone 

Female 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0. 0 

0 23 54 51 31 

0 6 0 0 0 

0 0 0 0 0 

1 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 25 32 21 13 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

7 12 5 8 7 

3 3 6 1 1 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 2 0 

9 14 9 10 1 

0 0 0 0 1 
30 Skin Paint Studies, NTP TR 441 

those in the vehicle controls. In Swiss (CD-l@) mice, those in the vehicle controls (Table 6). 

tumors in protocol control groups were similar to TPA protocol controls were significantly greater than 

In B6C3Fl and SENCAR mice, the incidences of skin the incidences of skin tumors in male and female 

Tumor Response in Protocol Controls 

in the Comparative Initiation/Promotion Skin Paint Studies: Protocol Controls for Study Design Aa 

Incidences of Skin Tumors in B6C3FlMice, Swiss (CD-1") Mice, and SENCAR Mice 

TABLE 6 

B6C3Fl Swiss (CD-1") SENCAR 

Acetone/Acetoneb 

AcetonelAcetone 

Male 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 Kg DMBNAcetone 

0.25 pg DMBNAcetone 

on0 

C - 

on0 

on0 

1/30 

on0 

on0 

20 mg BPODO mg BPO on0 on0 on0 

AcetonelAcetone 

AcetonelAcetone 

Female 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

OB0 

on0 

0/30 

20 mg BPOm mg BPO on0 On0 on0 

4/30. 

- 

- 

on0 

* Significantly different (PSO.05) from the vehicle control group by logistic regression a Number of animals with tumors per number of animals with skin examined in-life. Incidences represent clinical observations of 

SENCAR restart group 

Dose level not administered to this strain 

Vehicle control for TPA restart (SENCAR mice) 

papilloma. 
Results 31 

Survival of all B6C3Fl groups was similar to that of 

Survival 

AND TPA PROMOTION 

DMBA INITIATION 

accordance with the moribund sacrifice policy. 

25 pg DMBA/"A were removed from the study in 

of the Swiss (CD-l@) and SENCAR mice receiving 

respective vehicle controls (Table 7). However, many 

0.25,2.5, or 25 pg DMBA was lower than that of the 

Swiss (CD-l@) and SENCAR mice initiated with 

the vehicle controls. Survival of male and female 

(Table 7). 

similar to those of the respective vehicle controls 

(CD-l@) mice in the DMBmA groups were 

Final mean body weights of B6C3Fl and Swiss 

Body Weights and Clinical Findings 

more frequent during the later weeks of the study. 

10 weeks of the study; incidences of ulcer became 

incidences of irritation were observed during the first 

ages of Swiss (CD-l@) and SENCAR mice with 

Swiss (CD-l@) mice. Generally, the highest percentanimals and appeared to occur at a later time than in 

SENCAR mice, irritation was observed in fewer 

be related to the concentration of DMBA. In 

(CD-l@) mice during week 3 and did not appear to 

and females in all three DMBqrrPA groups of Swiss 

the most ulcers. Irritation was first observed in males 

incidences of skin irritation, and SENCAR mice had 

B6C3Fl mice. Swiss (CD-l@) mice had the highest 

ulcer were seldom observed in male or female 

surface excavation and/or broken skin. Irritation and 

sore appearance. Ulcer was defined as localized skin 

(Table 8). Irritation was defined as inflamed skin or 

receiving DMBA/"A were irritation and ulcer 

The primary clinical findings recorded for mice 
32 Skin Paint Studies, NTP TR 441 

in the Comparative Initiatioflromotion Skin Paint Studies: DMBA Initiation and TPA Promotion 

Survival and Body Weights of B6C3Fl Mice, Swiss (CD-la) Mice, and SENCAR Mice 

TABLE 7

to Controls (%) 

Relative 

Final Weight 

Initiator/Promoter Survivala Final Mean Body Weightb (g) 

50 pg DMBNS pg TPA 

25 pg DMBNS pg TPA 

2.5 pg DMBN5 pg TPA 

5 pg TPN5 pg TPA 

AcetoneJAcetone 

Male 

B6C3Fl 

50 pg DMBNS pg TPA 

25 pg DMBN5 pg TPA 

2.5 pg DMBN5 pg TPA 

5 pg TPN5 fig TPA 

AcetonelAcetone 

Female 

25 pg DMBN5 kg TPA 

2.5 pg DMBN5 pg TPA 

0.25 pg DMBN5 pg TPA 

5 pg TPN5 pg TPA 

AcetonelAcetone 

Male 

Swiss (CD-1") 

25 pg DMBN5 pg TPA 

2.5 pg DMBN5 pg TPA 

0.25 pg DMBN5 pg PA 

5 pg TPN5 pg TPA 

AcetonelAcetone 

Female 

25 pg DMBNI pg TPA 

2.5 pg DMBN1 pg TPA 

0.25 pg DMBM pg TPA 

1 pg TPN1 pg TPA 

AcetonelAcetone 

Male 

SENCAR 

25 pg DMBN1 pg TPA 

2.5 pg DMBM pg TPA 

0.25 pg DMBM pg TPA 

1 pg TPN1 pg TPA 

AcetonelAcetone 

Female 

26/30 

29/30 

3MO 

29/30 

26/30 

27/30 

28/30 

30/30 

30/30 

3OBO 

15BO** 

15/30** 

20/30 

21/30 

27/30 

16/30** 

19/30' 

22/30 

23/30 

27/30 

29/30 

li30** 

2/30* * 

6/30** 

9/30* * 

1/30** 

6/30* * 

8/30* * 

10/30** 

27/30 

46.5 f 0.8 

46.9 f 0.8 

45.7 ' 0.8 

46.8 f 0.8 

48.4 f 0.6 

42.1 * 1.1 

41.7 f 1.1 

42.1 f 0.7 

42.0 f 0.9 

44.4 f 0.9 

45.1 f l.7A 

45.1 f l.2A 

48.6 f 1.4 

47.5 f 1.4 

50.0 k 1.3 

38.4 f 1.3 

38.6 k 0.7 

38.2 k 0.9 

38.2 f 0.8 

40.1 f 1.5 

38.2 

46.5 f 1.3 

48.6 f 3.7 

48.8 f 2.8 

51.7 f 1.4 

39.2 

39.1 f 1.5 

47.2 f 2.9 

44.1 ? 2.6 

45.7 k 1.7 

96 

97 

94 

97 

95 

94 

95 

94 

90 

90 

97 

95 

96 

96 

95 

95 

74 

90 

94 

94 

86 

86 

103 

97 

a Number of animals survivinglnumber initially in group. Subsequent calculations are based on animals surviving to the end of the 

A Significantly different (PSO.05) from the vehicle control group by Dunnett's test 

** Significantly different (PSO.01) from the vehicle control group by life table pairwise comparison 

* Significantly different (PSO.05) from the vehicle control group by life table pairwise comparison 

Weights are given as mean f standard error. No standard errom were calculated for groups with high mortality. 

study. 
Results 33 

DMBA Initiation and TPA Promotion 

and SENCAR Mice in the Comparative InitiationPromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3Fl Mice, Swiss (CD-l@) Mice, 

TABLE 8 

Irritation Ulcer 

Week of Study 1-10 11-20 21-30 31-40 41-50 1-10 11-20 21-30 31-40 41-50 

50 pg DMBNS pg PA 

25 pg DMBNS pg 'PA 

2.5 pg DMBN5 pg TPA 

B6C3Fl 

Female 

25 pg DMBN5 pg 'PA 

2.5 pg DMBN5 pg PA 

0.25 pg DMBN5 pg TPA 

Swiss (CD-l@) 

25 pg DMBN1 pg TPA 

2.5 pg DMBN1 pg PA 

0.25 pg DMBAll pg TPA 

SENCAR 

0 2 0 1 1 

0 2 1 1 4 

0 3 1 1 2 

30 21 21 31 25 

26 33 13 5 11 

20 18 10 7 11 

I 14 6 4 0 

I 9 4 4 2 

6 5 4 6 0 

0 1 0 0 0 

0 0 1 4 10 

0 0 0 2 6 

26 13 5 3 9 

22 5 3 2 4 

30 21 19 29 32 

4 5 2 3 0 

4 7 4 3 0 

4 9 10 8 1 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

9 12 5 4 8 

1 5 16 20 22 

1 3 6 22 30 

0 22 32 48 24 

1 28 51 62 66 

0 15 41 63 61 

0 0 0 0 0 

0 0 0 0 3 

0 0 0 0 2 

0 8 11 7 0 

0 1 7 5 13 

0 0 2 10 11 

0 16 36 42 22 

0 9 30 41 56 

0 8 44 39 44 
34 Skin Paint Studies, NTP TR 441 

developed skin papillomas is given in Table 9. 

tive count. The number of mice per group that 

grooming. No mice were removed from the cumulamay have regressed or been removed by mechanical 

maintained for this study. In addition, some tumors 

because of the aggressive moribund sacrifice policy 

1-year study. In some groups, survival was reduced 

developed skin papillomas over the course of the 

displayed as the cumulative percentage of mice that 

are shown in Figures 1 and 2. The response is 

of DMBA followed by repeated applications of TPA 

mouse strains initiated with different concentrations 

tumors per mouse. The responses of the three 

appearance of the first tumor, and the number of 

group that developed tumors, the group mean time to 

tumor development: the number of animals in each 

sensitivity of each mouse strain to chemical-induced 

Three parameters were used to measure the skin 

Tumor Response 

ing concentration of the initiator DMBA in males 

Incidences of skin papillomas increased with increasrespective promoter controls (Table 9). 

all mice receiving DMBA was greater than that of the 

controls indicated that the skin papilloma response of 

initiating concentrations and the TPA/TPA promoter 

sons within strains between the different DMBA 

and females of each mouse strain. Painvise compariSENCAR mice. 

was five times greater for Swiss (CD-l@) than for 

even though the concentration of the TPA promoter 

(CD-la) and SENCAR mice were remarkably similar 

papilloma in the three DMBmA groups for Swiss 

strains. The mean times to first appearance of 

concentration administered to the other mouse 

DMBA concentration that was twice as great as the 

of the fact that B6C3F, mice received an initiating 

than for Swiss (CD-l@) and SENCAR mice in spite 

appearance of papilloma was greater for B6C3Fl mice 

mas) is shown in Table 9. The mean time to the first 

loma (based on the animals that developed papilloThe mean time to the appearance of the first papil- 
Results 35 

100 

75 

m 

25 

0 

1w 

0 

loa 

rs 

2s 

0 

FIGURE 1

B6C3F1 

SENCAR 

10 20 30 40 50 I 

Week on Test 

100 

75 

SO 

2s 

0 

Followed by 5 fig [B6C3Fl and Swiss (CD-la)] or 1 Kg (SENCAR) TPA Promotion 

Tumor Response of Male Mice of the Three Strains Initiated with Different Concentrations of DMBA 
36 Skin Paint Studies, NTP TR 441 

loo 

50 

% 

0 

100 

0 

100 

75 

50 

0 

FIGURE 2 

10 so 30 40 50 I 

Week on Test 

loo 

rs 

50 

0 

loo 

75 

50 

?5 

0 

100 

75 

50 

25 

0 

Followed by 5 pg [B6C3Fl and Swiss (CD-le)] or 1 pg (SENCAR) TPA Promotion 

Tumor Response of Female Mice of the Three Strains Initiated with Different Concentrations of DMBA 
Results 37 

DMBA Initiation and TPA Promotion 

in the Comparative Initiatioflromotion Skin Paint Studies: 

Papilloma Response in B6C3Fl Mice, Swiss (CD-lm) Mice, and SENCAR Mice 

TABLE 9 

DMBN 5 pg TPN 2.5 pg DMBN 25 pg DMBN 50 pg DMBN 

B6C3Fl Acetone 5 pg TPA 5 pg TPA 5 pg TPA 5 pg TPA 

Life table testd 

Life table test' 

Overall rateb 

Mean weeks to first papillomaa 

Male 

on0 

e - - 

P<O.OOl P<O.OOl 

P <0.001 

on0 lono 

35.4 f 3.21 

P<O.OOl 

P < 0.001 

12/30 

27.6 f 3.82 

P<O.OOl 

P <0.001 

22/30 

32.5 f 2.46 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female - - 

P<O.OOl P=O.118 

P=O.124 

on0 3/30 

36.7 f 3.93 

on0 

P=O.O07 

P=O.O07 

7/30 

41.0 f 4.40 

P<O.OOl 

P<O.OOl 

16/30 

40.8 f 2.47 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Male - f 

P<O.OOl P < 0.001 

P<O.OOl 

5/30 18/30 

34.6 f 6.38 27.7 2 3.04 

P<O.OOl 

P<O.OOl 

27/30 

15.4 & 0.79 

P<O.OOl 

P < 0.001 

28/30 

17.6 f 1.78 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female - 

P<O.OOl P<O.OOl 

P<O.OOl 

4/30 18/30 

42.3 f 2.98 26.5 f 2.95 

on0 

PCO.001 

P<O.OOl 

26/30 

18.0 & 1.61 

P<O.OOl 

PiO.001 

29/30 

16.0 f 1.37 

Acetone 1 pg TPA 1 pg TPA 1 pg TPA 1 pg TPA 

DMBN 0.25 pg DMBN 2.5 pg 25 fig DMBN 

SENCAR 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Male - 

P<O.OOl P <0.001 

P<O.OOl 

2/30 12/30 

16.0 f 5.00 17.2 f 1.93 

on0 

P<O.OOl 

P < 0.001 

19/30 

15.5 f 1.15 

P<O.OOl 

P<O.OOl 

24/30 

14.5 f 1.35 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female - - 

P<O.OOl P co.001 

P<O.OOl 

OB0 13/30 

23.5 f 2.79 

on0 

P<O.OOl 

PCO.001 

27/30 

15.9 f 0.86 

P<O.OOl 

P<O.O01 

29/30 

14.4 f 0.99 

(continued) 
38 Skin Paint Studies, NTP TR 441 

DMBA Initiation and TPA Promotion (continued) 

in the Comparative Initiatioflromotion Skin Paint Studies: 

Papilloma Response in B6C3Fl Mice, Swiss (CD-l@) Mice, and SENCAR Mice 

TABLE 9 

a Mean f standard error 

groups. 

SENCAR] group incidences are the P values corresponding to pairwise comparisons between the TPAKF'A group and those 

50 pg DMBA/TPA (B6C3Fl) or the 0.25 pg DMB-A, 2.5 pg DMB-A, and 25 pg DMBAEF'A [Swiss (CD-le) and 

In the TP-A columns are the P values associated with the trend test. Beneath the 2.5 pg DMB-A, 25 pg DMB-A, and 

pairwise comparisons between the corresponding DMBNAcetone group and those groups. 

2.5 pg DMB-A, and 25 pg DMB-A [Swiss (CD-la) and SENCAR] group incidences are the P values corresponding to 

Beneath the 2.5 pg DMB-A, 25 pg DMBAfI'PA, and 50 pg DMB-A (B6C3Fl) or the 0.25 pg DMBmPA, 

Number of animals with tumor per number of animals with skin examined in-life 

25 pg DMBNAcetone groups had overall rates of 0/30. 

The 0.25 pg DMBNAcetone group had an overall rate of 1/30 and mean weeks to first papilloma of 50.0; the 2.5 and 

e Not applicable; no papillomas obsewed in animal group unless otherwise noted 
Results 39 

The average number of papillomas per mouse was presented in Table 10. The data used to calculate 

calculated two ways. In the first method, the total tumor multiplicity were collected at 10-week intervals 

number of tumors in each group was divided by the over the course of the study. Swiss (CD-la) and 

number of animals that developed tumors. In the SENCAR mice had more tumors per mouse than did 

second method, the total number of tumors in each B6C3Fl mice, and the tumor multiplicity increased 

group was divided by the total number of animals in with increasing initiating DMBA concentration, in a 

each group. The values from both methods are significant trend. 

DMBA Initiation and TPA Promotion 

Papillomas Observed During the Comparative Initiation/Promotion Skin Paint Studies: 

TABLE 10 

Week of Study 10 20 30 40 so 

50 pg DMBNS pg PA 

25 pg DMBNS pg PA 

2.5 pg DMBN5 pg TPA 

Male 

B6C3Fl 

50 pg DMBA/5 pg "A 

25 pg DMBNS pg TPA 

2.5 pg DMBA/S pg PA 

Female 

25 pg DMBNS pg TPA 

2.5 pg DMBA/5 pg TPA 

0.25 pg DMBN5 pg TPA 

Male 

Swiss (CD-le) 

25 pg DMBN5 pg TPA 

2.5 pg DMBN5 pg TPA 

0.25 pg DMBN5 pg TPA 

Female 

25 pg DMBM Irg TPA 

2.5 pg DMBM pg PA 

0.25 pg DMBNI pg PA 

Male 

SENCAR 

25 pg DMBA/l pg TPA 

2.5 pg DMBM kg 

0.25 pg DMBN1 pg "A 

Female 

oa ob 

00 

00 

00 

00 

00 

2.25 0.10 

1.00 0 

00 

1.00 0.03 

00 

00 

1.00 07

1.00 03

00 

2.00 013

2.00 0.14

00 

1.00 0.03 1.00 0.03 1.33 0.27 1.75 0.47 

2.00 0.33 1.88 0.50 1.90 0.63 2.50 0.69 

1.00 0.14 1.50 0.41 2.13 1.10 2.53 1.46 

00 1.00 0.03 2.00 0.13 1.67 0.17 

1.00 0.03 1.00 0.03 1.00 0.10 2.00 0.28 

1.00 0.03 1.50 0.10 1.29 0.32 1.75 0.78 

2.14 0.05 2.13 0.63 1.90 0.79 1.70 0.75 

3.72 3.07A 4.44 3.83A 3.19 2.88A 2.69 1.94A 

5.50 3.87A 4.88 4.43A 4.28 3.19 2.60 1.88 

2.29 0.53 3.14 0.73 2.27 0.93 3.14 1.00 

3.90 2.33A 4.10 2.83A 3.47 2.54A 3.15 2.05A 

6.61 5.07A 7.04 6.52A 4.91 4.70A 3.87 3.5OA 

1.73 0.82 2.38 1.19 2.17 1.00 1.00 0.50 

3.82 1.68A' 3.50 2.15A 2.75 1.3W 1.00 0.50 

5.74 1.62A' 5.64 2.89A' 4.60 2.30 1.00 0.50 

1.67 0.40 1.67 0:48 1.88 0.94 1.67 25

4.24 3.07A 3.56 2.33A 3.18 1.94A 2.00 1.63A 

7.17 6.38A 6.53 4.4S 5.86 4.70A 00 

Average number of papillomas expressed as the total number of papillomas/number of mice surviving 

a Average number of papillomas wpressed as the total number of papillomas/number of mice with papillomas 

Significantly different (PSO.05) from similarly treated Swiss (CD-le) groups by the Mann-Whitney U test 

A Significantly different (PSO.05) from similarly treated B6C3Fl groups by the Mann-Whitney U test 
40 Skin Paint Studies, NTP TR 441 

controls (Table 11). 

initiating dose was lower than that of the vehicle 

male SENCAR mice receiving the 25 pg DMBA 

that of the respective vehicle controls. Survival of 

(CD-1") and female SENCAR mice was similar to 

Survival of male and female B6C3Fl and Swiss 

Survival 

AND BPO PROMOTION 

DMBA INITIATION 

(Table 11). 

groups were similar to those of the vehicle controls 

Final mean body weights of DMBA/BPO study 

Body Weights and Clinical Findings 

and in females during weeks 21 through 30. 

B6C3Fl males appeared during weeks 11 through 20 

for male SENCAR mice. Signs of irritation in the 

(CD-l@) and female SENCAR mice and in week 5 

first noted in week 3 for male and female Swiss 

male than in female mice. Signs of irritation were 

general, these signs were more frequently observed in 

irritation or more than 10% for ulcers (Table 12). In 

these signs never averaged more than 15% for 

ulcer, were minimal. Over the course of the study 

Clinical signs of chemical exposure, irritation and 
Results 

in the Comparative Initiatioflromotion Skin Paint Studies: DMBA Initiation and BPO Promotion 

Survival and Body Weights of B6C3Fl Mice, Swiss (CD-la) Mice, and SENCAR Mice 

TABLE 11 

to Controls (%) 

Relative 

Final Weight 

Initiator/Promoter Survivala Final Mean Body Weightb (9) 

Male 

B6C3Fl 

25 pg DMBADO mg BPO 

2.5 pg DMBAnO mg BPO 

20 mg BP0/20 mg BPO 

AcetonelAcetone 

Female 

25 Kg DMBAnO mg BPO 

2.5 pg DMBAnO mg BPO 

20 mg BPOnO mg BPO 

AcetonelAcetone 

Male 

Swiss (CD-lm) 

25 pg DMB-0 mg BPO 

2.5 pg DMBWO mg BPO 

20 mg BPOnO mg BPO 

AcetoneJAcetone 

Female 

25 pg DMBA/20 mg BPO 

2.5 pg DMBA/2O mg BPO 

20 rng BPODO mg BPO 

Acetone/Acetone 

Male 

SENCAR 

25 pg DMBAnO mg BPO 

2.5 fig DMBADO mg BPO 

20 mg BPODO mg BPO 

AcetonelAcetone 

Female 

25 Kg DMB-0 mg BPO 

2.5 pg DMBADO mg BPO 

20 mg BPOnO mg BPO 

AcetonelAcetone 

30/30 

27/30 

30/30 

26/30 

29/30 

28/30 

27/30 

3OPO 

24/30 

27/30 

25/30 

27/30 

30130 

28/30 

30/30 

27/30 

17/30** 

23/30 

23/30 

28/30 

23/30 

21/30 

28/30 

27/30 

46.5 f 0.9 

47.0 rt 0.7 

46.2 f 0.8 

48.4 f 0.6 

43.1 f 0.8 

41.9 f 1.0 

43.1 f 0.9 

44.4 f 0.9 

50.7 f 1.4 

50.0 * 1.3 

51.6 f 1.3 

50.0 ? 1.3 

42.6 rt 1.3 

40.4 & 1.0 

40.8 1.1 

40.1 & 1.5 

46.1 f 1.6A 

49.6 f 1.5 

50.7 f 1.4 

51.7 rt 1.3 

41.9 f 1.0 

40.8 f 1.2 

41.1 rt 1.2 

44.5 f 1.4 

96 

97 

95 

97 

94 

97 

101 

100 

103 

106 

101 

102 

89 

96 

98 

94 

92 

92 

a Number of animals survivinghumber initially in group. Subsequent calculations are based on animals surviving to the end of the study. 

A Significantly different (PSO.05) from the vehicle control group by Dunnett’s test 

** Significantly different (P~0.01) from the vehicle control group by life table pairwise comparison 

Weights are given as mean +- standard error. 
42 Skin Paint Studies, NTP TR 441 

DMBA Initiation and BPO Promotion 

and SENCAR Mice in the Comparative InitiatiodPromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3F, Mice, Swiss (CD-l@) Mice, 

TABLE 12 

Irritation Ulcer 

Week of Study 1-10 11-20 21-30 31-40 41-50 1-10 11-20 21-30 31-40 41-50 

25 pg DMBADO mg BPO 

2.5 pg DMBAnO mg BPO 

B6C3Fl 

Male 

25 pg DMBAnO mg BPO 

2.5 pg DMBADO mg BPO 

Swiss (CD-lB) 

25 pg DMBADO mg BPO 

2.5 pg DMBADO mg BPO 

SENCAR 

25 pg DMBAnO mg BPO 

2.5 pg DMBAnO mg BPO 

B6C3Fl 

Female 

Swiss (CD-l@) 

25 fig DMBAnO mg BPO 

2.5 pg DMBAnO mg BPO 

25 pg DMBA/20 mg BPO 

2.5 pg DMBA120 mg BPO 

SENCAR 

0 3 6 4 7 

0 1 6 7 10 

3 3 5 10 7 

4 1 4 13 13 

2 7 10 1 3 

4 14 15 4 9 

0 0 3 7 10 

0 0 0 0 0 

1 0 1 1 1 

1 0 0 1 1 

1 2 9 0 2 

3 2 7 0 0 

0 0 2 7 4 

0 1 3 5 4 

0 2 1 5 8 

1 2 1 0 1 

0 0 0 3 5 

1 2 4 10 6 

0 0 1 8 10 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 0 

0 0 0 0 1 

0 2 0 0 2 
Results 43 

Tumor Response skin papillomas over the course of the 1-year study. 

The response of mice receiving 2.5 or 25 pg DMBA Some tumors may have regressed or been removed by 

initiation followed by repeated applications of BPO mechanical grooming. No mice were removed from 

is shown in Figure 3. The response is displayed as the cumulative count. The number of mice per group 

the cumulative percentage of mice that developed that developed skin papillomas is given in Table 13. 

DMBA Initiation and BPO Promotion 

in. the Comparative Initiation/Promotion Skin Paint Studies: 

Papilloma Response in B6C3F, Mice, Swiss (CD-la) Mice, and.SENCAR Mice 

TABLE 13 

DMBA/ 20 mg BPO/ 2.5 kg DMBA/ 25 kg DMBA/ 

Acetone 20 mg BPO 20 mg BPO 20 mg BPO 

Life table testd 

Life table test' 

overall rateb 

Mean weeks to first papillomaa 

Male 

B6C3F, 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female 

Life table test 

Life table test 

Overall rate 

Mean weeks to first. papilloma 

Male 

Swiss (CD-1@) 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female 

0/30 

e - 

- 

on0 

- 

on0 

P=O.551 

- 

on0 

P=O.601 

P=O.O02 

- 

on0 

P=O.OlO 

P=O.493 

P=O.493 

1/30 

48.0 

P=O.O65 

P=O.O56 

4/30 

51.0 k 0.58 

P=O.515 

P = 0.486 

1/30 

52.0 

P=O.486 

P=O.493 

1/30 

51.0 

P=O.500 

P=O.507 

1/30 

39.0 

P=O.248 

P=O.229 

2/30 

44.0 k 7.00 

P=O.O12 

P=O.O09 

6/30 

47.0 k 2.25 

P=O.O31 

P=O.O34 

5/30 

39.2 2 4.07 

(continued) 
44 Skin Paint Studies, NTP TR 441 

DMBA Initiation and BPO Promotion (continued) 

in the Comparative Initiatioflromotion Skin Paint Studies: 

Papilloma Response in B6C3Ft Mice, Swiss (CD-1@) Mice, and SENCAR Mice 

TABLE 13 

Acetone 20 mg BPO 20 mg BPO 20 mg BPO 

DMBA/ 20 mg BPO/ 2.5 pg DMBA/ 25 pg DMBA/ 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma - 

Male 

SENCAR 

on0 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma - 

Female 

on0 

- 

P <0.001 

on0 

P < 0.001 

P<O.OOl 

20BO 

31.0 zk 2.25 

P<O.OOl 

P <0.001 

22130 

31.0 +. 2.46 

- 

P < 0.001 

P<O.OOl 

22/30 

31.2 * 2.04 

P <0.001 

OB0 

P<O.ool 

P<O.OOl 

20/30 

33.6 2 1.88 

a Mean & standard deviation 

20 mg BPODO mg BPO group and those groups. 

and 25 pg DMBMO mg BPO group incidences are the P values corresponding to pairwise comparisons between the 

In the 20 mg BPODO mg BPO columns are the P values associated with the trend test. Beneath the 2.5 pg DMBMO mg BPO 

comparisons between the corresponding DMBNAcetone group and those groups. 

Beneath the 2.5 pg DMBMO mg BPO and 25 pg DMBAf20 mg BPO group incidences are the P values corresponding to pairwise 

Number of animals with tumor per number of animals with skin examined in-life 

e Not applicable; no papillomas observed in animal group 

not statistically significant. 

greater than those in controls, but the increase was 

25 pg DMBA, the incidences in B6C3Fl mice were 

male and female SENCAR mice initiated with 2.5 or 

(CD-l@) mice initiated with 25 pg DMBA and in 

than those in controls in male and female Swiss 

The incidences of papilloma were significantly greater 

papilloma (based on the animals that developed 

The mean time to the appearance of the first 

(CD-l@) mice. 

DMBA male B6C3Fl and male and female Swiss 

there were incidences of only one tumor in 2.5 pg 

initiating concentrations of 2.5 pg DMBA, although 

time in the study than did those mice that received 

DMBA appeared to develop tumors at an earlier 

that received initiating concentrations of 25 pg 

weeks 31 to 34. B6C3Fl and Swiss (CD-l@) mice 

and the mean time to first papilloma appearance was 

appeared in SENCAR mice between weeks 9 and 10 

papillomas) is shown in Table 13. Papillomas first 
Results 45 

Week on Test 

Followed by 20 mg BPO Promotion 

Tumor Response of the Three Mouse Strains Initiated with 2.5 or 25 Kg DMBA 

FIGURE 3 
46 Skin Paint Studies, NTP TR 441 

The average number of papillomas per mouse was mice receiving initiating concentrations of 2.5 pg and 

calculated two ways as described previously, and these 25 pg DMBA averaged similar numbers of papillomas 

averages are presented in Table 14. The data used to per mouse. SENCAR mice averaged more papillocalculate tumor multiplicity were collected at10-week mas per mouse than did B6C3Fl or Swiss (CD-l@) 

intervals over the course of the study. SENCAR mice. 

DMBA Initiation and BPO Promotion 

Papillomas Observed During the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE 14 

Week of Study 10 20 30 40 50 

25 pg DMBAnO mg BPO 

2.5 pg DMBAnO mg BPO 

Male 

B6C3Fl 

25 pg DMBAnO mg BPO 

2.5 pg DMBAn0 mg BPO 

Female 

25 pg DMBAn0 mg BPO 

2.5 pg DMBAnO mg BPO 

Male 

Swiss (CD-l@) 

25 pg DMBAn0 mg BPO 

2.5 pg DMBAnO mg BPO 

Female 

25 pg DMBAnO mg BPO 

2.5 pg DMBAn0 mg BPO 

Male 

SENCAR 

25 pg DMB-0 mg BPO 

2.5 pg DMBAnO mg BPO 

Female 

Oa 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Ob 

0 0 

0 

0 0 

0 0 

0 0 

0 0 

0 0 

0 0 

0 3.00 

0 1.43 

0 1.50 

0 0 

0 

0 

00 

00 00 1.00 0.04 

1.00 0.03 00 

0 

0 

00 

00 00 1.00 0.07 

1.00 0.03 1.00 0.03 

0 

0 

00 

00 00 00 

1.00 0.04 1.00 0.08 

0 

0 00 00 00 

1.00 0.03 1.50 0.10 1.50 0.20 

0.31 4.00 l.24AT 2.21 1.07Ar 2.73 1.64Ar 

0.33Ar 3.00 1.03A’ 3.60 1.3PT 3.20 1.68Ar 

0.20rr 3.20 l.lOAr 3.06 1.86Ar 1.69 1 .23Ar 

0 1.88 0.54Av 3.07 1.70Ar 3.00 1.96Ar 

Average number of papillomas expressed as the total number of papillomas/number of mice surviving 

a Average number of papillomas expressed as the total number of papillomas/number of mice with papillomas 

Significantly different (PSO.05) from similarly treated Swiss (CD-l@) groups by the Mann-Whitney U test 

A Significantly different (PSO.05) from similarly treated B6C3Fl groups by the Mann-Whitney U test 
Results 47 

COMPARISON OF THE MOUSE STRAIN 

DMBAITPA: The response of the three strains of 

AS A PROMOTER 

AS AN INITIATOR AND TPA OR BPO 

similar in Swiss (CD-l@) and SENCAR mice, even 

doses (Table 15). The incidences of papilloma were 

2.5 and 25 pg DMBA were used as the initiation 

significantly greater than those in B6C3Fl mice when 

and female Swiss (CD-l@) and SENCAR mice were 

Figures 4 and 5. The incidences of papilloma in male 

PA promotion over the 1-year study is shown in 

(SENCAR) or 5 pg [B6C3Fl and Swiss (CD-l@)] 

mice to 2.5 or 25 pg DMBA initiation and 1 pg 

RESPONSE TO DMBA 

(1 

trations of TPA (5 pg) than did the SENCAR mice 

though Swiss (CD-l@) mice received higher concennumbers of papillomas than did B6C3Fl mice. 

appearance of the first papilloma, and higher average 

papilloma incidences, shorter group mean time to the 

Swiss (CD-l@) and SENCAR mice had higher 

lent initiating concentrations of DMBA, 

lowest dbncentration (0.25 pg) of DMBA. At equivaSwiss (CD-la) and SENCAR mice receiving the 

concentration of DMBA (50 pg) was most similar to 

response of B6C3Fl mice receiving the highest 

mice are summarized in Table 16. The papilloma 

The in-life papilloma observations for DMBmA 
E g 1 

Papillomas 

Cumulative Percent Mice with 

I I I 

0 E 0 

VI UI 

-l ii 

Cumulative Percent Mice with 

.Papillomas . - 

0 .' ., I 

.. .. <' ,g I 

: VI 

* 

01 

0) 

I I 1 

0 E 8 -. , 
e F9 2 

Papillomas 

Cumulative Percent Mice with 

0 c: !3 -J 

VI 

8 

Papillomas 

Cumulative Percent Mice with 

VI 

0 G? 

I I I 

0 VI 

Io :: -. 

VI 

-4 0 

0 

W 

P 
50 

~ 

Skin Paint Studies, NTP TR 441 

DMBA Initiation and TPA Promotion 

in the Comparative Initiatioflromotion Skin Paint Studies: 

Comparisons of Papilloma Response Between B6C3Fl Mice, Swiss (CD-l@) Mice, and SENCAR Mice 

TABLE 15 

B6C3Fl Swiss (CD-l@) SENCAR 

Male 

Life table testd 

Life table test' 

Overall rateb 

2.5 bg DMBA~~PA~ 

Life table test 

Life table test 

Overall rate 

25 pg DMBAlrPA 

Life table test 

Life table test 

Overall rate 

2.5 pg DMBA/TPA 

Female 

Life table test 

Life table test 

Overall rate 

25 pg DMBA/TPA 

10/30 

12/30 

3/30 

7/30 

PCO.001 

27/30 

P < 0.001 

28/30 

PCO.001 

26/30 

PCO.001 

29/30 

P=0.106N 

P c 0.001 

19/30 

P=0.484N 

P co.001 

=BO 

P=O.119 

PCO.001 

27/30 

P-0.208 

PcO.001 

29/30 

' P values correspond to painvise comparisons with B6C3Fl mice. 

a B6C3Fl mice and Swiss (CD-l@) mice received 5 pg "A; SENCAR mice received 1 pg TPA. 

P values correspond to painvise comparisons Swiss (CD-l@) mice. A lower incidence in a dose group is indicated by N. 

Number of animals with tumor per number of animals with skin examined in-life 
Results 51 

Overview of DMBNTPA Sensitivity in the Comparative InitiationPromotion Skin Paint Studies 

TABLE 16 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors Total Micea Mice with Tumorsb (Week) 

50 pg DMBNS pg TPA 

25 pg DMBN5 pg TPA 

2.5 pg DMBN5 pg TPA 

Male 

B6C3F, 

50 pg DMBNS pg TPA 

25 pg DMBNS pg TPA 

2.5 pg DMBN5 pg TPA 

Female 

22/30 

12/30 

lO/30 

16/30 

7/30 

3/30 

25 pg DMBN5 pg TPA 

2.5 pg DMBN5 pg TPA 

0.25 pg DMBNS pg TPA 

Male 

Swiss (CD-l@) 

25 pg DMBN5 pg TPA 

2.5 pg DMBN5 fig TPA 

0.25 pg DMBNS pg TPA 

Female 

28/30 

27/30 

18/30 

29/30 

25/30 

18/30 

25 pg DMBM pg TPA 

2.5 pg DMBM pg TPA 

0.25 pg DMBN1 pg TPA 

Male 

SENCAR 

25 pg DMBfVl pg PA 

2.5 pg DMBM fig PA 

0.25 pg DMBN1 pg TPA 

Female 

2/30 

19/30 

12/30 

29/30 

27/30 

13/30 

32.5 

27.6 

35.4 

40.8 

41.0 

36.7 

17.6 

15.4 

27.7 

16.0 

18.0 

26.5 

14.5 

15.5 

17.2 

14.4 

15.9 

23.5 

1.95 

1.13 

0.57 

1.10 

0.37 

0.27 

5.60 

4.63 

1.07 

7.10 

3.77 

1.43 

6.77 

3.03 

1.10 

9.23 

5.23 

0.87 

2.66 

2.83 

1.70 

2.06 

1.57 

2.67 

6.00 

5.15 

1.78 

7.34 

4.35 

2.39 

8.46 

4.79 

2.75 

9.55 

5.81 

2.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

tumors in each group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed 
52 Skin Paint Studies, NTP TR 441 

in both male and female B6C3Fl and Swiss (CD-l@) 

tions of DMBA. The papilloma response was similar 

Swiss (CD-l@) mice for both initiation concentrasignificantly greater than that in B6C3F, and 

The incidence of papilloma in SENCAR mice was 

the most responsive to this combination of chemicals. 

are shown in Figures 6 and 7. SENCAR mice were 

common) and BPO promotion over the 1-year study 

mice to 2.5 or 25 pg DMBA initiation (doses in 

DMBAIBPO: The responses of the three strains of 

groups receiving 25 pg DMBA (Table 17). 

male Swiss (CD-l@) mice than in B6C3Fl males for 

mice that received 2.5 pg DMBA, but was higher in 

B6C3Fl or Swiss (CD-l@) mice. 

greater average number of tumors than did the 

shorter mean group time to first papilloma, and a 

higher papilloma incidence, SENCAR mice had 

mice are summarized in Table 18. In addition to a 

The in-life papilloma observations for DMBA/BPO 
Results 53 

--O- SENCAR 

* swiss I 

25 pg DMBA 

I I I I 1 

10 20 30 40 50 

Week on Test 

Followed by 20 mg BPO Promotion 

Tumor Response of Male Mice of the Three Strains Initiated with 2.5 or 25 pg DMBA 

FIGURE 6 

100 

75 

50 

25 

0 
54 Skin Paint Studies, NTP TR 441 

-13- BbC3F1 

-0- Swiss 

.--+-- SENCAR 

D 

0 

I 1 I I I 

10 20 30 40 50 

2 

0 

Week on Test 

Week on Test 

100 

75 

so 

25 

0 

Followed by 20 mg BPO Promotion 

Tumor Response of Female Mice of the Three Strains Initiated with 2.5 or 25 fig DMBA 

FIGURE 7 
Results 55 

DMBA Initiation and BPO Promotion 

in the Comparative Initiatioflromotion Skin Paint Studies: 

Comparisons of Papilloma Response Between B6C3Fl Mice, Swiss (CD-l@) Mice, and SENCAR Mice 

TABLE 17 

B6C3F, Swiss (CD-la) SENCAR 

Life table test' 

Life table testb 

Overall ratea 

2.5 kg DMBAnO mg BPO 

Male 

Life table test 

Life table test 

Overall rate 

25 pg DMBAnO mg BPO 

Life table test 

Life table test 

Overall rate 

2.5 pg DMBAnO mg BPO 

Female 

Life table test 

Life table test 

Overall rate 

25 lg DMBMO mg BPO 

1/30 

1/30 

4/30 

2/30 

P=O.757 

1/30 

P=O.O32 

6/30 

P=0.175N 

1/30 

P=O.212 

5/30 

P<O.ool 

P<O.OOl 

m/30 

P < 0.001 

P < 0.001 

22/30 

P<O.OOl 

P<O.OOl 

22/30 

P<O.ool 

P<O.OOl 

2OBO 

' P values correspond to pairwise comparisons with Swiss (CD-le) mice. 

a Number of animals with tumor per number of animals with skin examined in-life 

P values correspond to painvise comparisons with B6C3Fl mice. A lower incidence in a dose group is indicated by N. 
56 Skin Paint Studies, NTP TR 441 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

with Tumors Total Micea Mice with Tumorsb (Week) 

25 pg DMBAnO mg BPO 

2.5 pg DMBm mg BPO 

Male 

B6C3Fl 

25 pg DMBAnO mg BPO 

2.5 pg DMBA/ZO mg BPO 

Female 

1/30 

1/30 

m 

4/30 

25 pg DMBm mg BPO 

2.5 pg DMBMO mg BPO 

Male 

Swiss (CD-la) 

6/30 

1/30 

25 pg DMBArU) mg BPO 

2.5 Fg DMBm mg BPO 

Female 

5/30 

1/30 

25 pg DMBm mg BPO 

2.5 pg DMBm mg BPO 

Male 

SENCAR 

25 pg DMBMO mg BPO 

2.5 pg DMBMB mg BPO 

Female 

22/30 

20i30 

20/30 

22/30 

39.0 

48.0 

44.0 

51.0 

47.0 

52.0 

39.2 

51.0 

31.0 

31.0 

33.6 

31.2 

0.03 

0.03 

0.07 

0.13 

0.20 

0.03 

0.30 

0.03 

2.77 

2.40 

2.50 

2.33 

1.00 

1.00 

1 .oo 

1 .oo 

1.00 

1.00 

1.80 

1.00 

3.77 

3.60 

3.75 

3.18 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

tumors in each group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed 
Results 57 

PROTOCOL CONTROL GROUPS 

STUDY DESIGN B 

without MNNG initiation (Table 2). 

1 ,ug/mouse in SENCAR] or BPO (20 mg/mouse) 

[5 pglmouse in B6C3Fl and Swiss (CD-l@) and 

received repeated applications of either TPA 

tions of the non-promoter acetone, and groups that 

500, or 1,000 pglmouse) followed by repeated applicacation of one of three concentrations of MNNG (100, 

alone, as well as groups that received a single applimice of each strain that received the acetone vehicle 

control groups included groups of male and female 

skin initiation/promotion model functioned. These 

were included as a measure of how well the mouse 

a number of initiator and promoter control groups 

PA and BPO as promoters. As in study design A, 

guanidine (MNNG) was used as the initiator with 

In study design B, N-methyl-N'-nitro-k-nitrosoby the non-promoter acetone was significantly lower 

Survival of mice receiving 1,000 pug MNNG followed 

Survival of Protocol Controls 

cantly lower than that of the vehicle controls. 

ing repeated applications of TF'A was also signifiSENCAR mice and male Swiss (CD-l@) mice receivvehicle controls. Survival of male and female 

acetone was significantly lower than that of the 

SENCAR mice receiving 500 pg MNNG followed by 

(Table 19). Survival of male Swiss (CD-l@) and 

mice of all strains except male B6C3Fl mice 

than that of the vehicle controls for male and female 

that of the vehicle controls. 

1,000 pg MNNG followed by acetone was lower than 

mean body weight of SENCAR males receiving 

lower than those of the vehicle controls. The final 

receiving repeated applications of TPA or BPO were 

final mean body weights of female B6C3Fl mice 

of the respective vehicle controls (Table 19). The 

mouse protocol control groups were similar to those 

Swiss (CD-l@), male B6C3Fl, and female SENCAR 

Final mean body weights of male and female 

Protocol Controls 

Body Weights and Clinical Findings in 

in the Comparative Initiation/Promotion Skin Paint Studies: Protocol Controls for Study Design B 

Survival and Body Weights of B6C3Fl Mice, Swiss (CD-1") Mice, and SENCAR Mice 

TABLE 19 

to Controls (%) 

Relative 

Final Weight 

Initiator/Prornoter Survivala Final Mean Body Weightb (9) 

Male 

B6C3F, 

AcetonelAcetone 30/30 

100 pg MNNG/Acetone 30/30 

500 pg MNNGlAcetone 29/30 

1,000 pg MNNG/Acetone 28/30 

5 fig TF'A/5 pg TPA 30/30 

20 mg BPOnO mg BPO 30/30 46.8 f 0.8 

48.1 k 0.6 

45.8 f 0.9 

47.6 +. 0.7 

41.9 f 0.5 

47.5 f 0.1 

99 

101 

96 

100 

101 

Female 

AcetonelAcetone 30/30 

100 pg MNNG/Acetone 30/30 

500 pg MNNG/Acetone 30/30 

1,000 pg MNNG/Acetone 23/30: 

5 pg TPN5 pg TPA 30130 

20 mg BPOm mg BPO 29/30 43.4 rt 0.9AA 

45.0 k 0.7A 

46.5 rt 1.1 

46.5 f 1.1 

41.3 k 1.2 

47.9 k 1.2 

91 

94 

97 

97 

99 

(continued) 
58 

~~ ~ 

Skin Paint Studies, NTP TR 441 

in the Comparative Initiatioflromotion Skin Paint Studies: Protocol Controls for Study Design B 

Survival and Body Weights of B6C3Fl Mice, Swiss (CD-lm) Mice, and SENCAR Mice 

TABLE 19 

(continued) 

to Controls (%) 

Relative 

Final Weight 

Initiator/Promoter Survival Final Mean Body Weight (g) 

Male 

Swiss (CD-l@) 

20 mg BPODO mg BPO 

5 fig TPN5 pg TPA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 yg MNNG/Acetone 

Acetone/Acetone 

Female 

20 mg BPODO mg BPO 

5 yg TPN5 pg TPA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

Acetone/Acetone 

Male 

SENCAR 

20 mg BP0/20 mg BPO 

1 kg TF" kg 'PA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

Acetone/AcetoneC 

AcetonelAcetone 

Female 

20 mg BPODO mg BPO 

1 pg TPM pg "PA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

Acetone/Acetone' 

Acetone/Acetone 

20/30 

17/30.; 

9/30* * 

17/30** 

28/30 

27/30 

29130 

23/30 

15/30** 

26130 

29/30 

27/30 

26/30 

9130; * 

7/30; * 

16/30°** 

23/30 

29/30 

29/30 

27/30 

10/30** 

11/30*; 

22BO 

30/30 

27/30 

26/30 

48.4 f 1.6 

48.1 f 1.4 

48.5 f 2.9 

51.6 f 1.6 

50.6 f 1.4 

50.7 f 1.3 

40.7 f 1.1 

37.4 & 1.3 

37.9 f 1.7 

39.8 2 1.0 

39.6 k 1.0 

39.0 f 1.1 

52.5 2 1.0 

48.8 -C 2.8 

45.5 f 2.2A 

48.6 f 1.5 

51.6 f 1.2 

51.7 2 1.4 

52.0 f 1.2 

46.2 f 1.8 

44.1 i- 2.6 

43.8 f 1.6 

42.6 i- 1.4 

44.3 f 1.3 

45.7 2 1.7 

44.9 f 1.4 

95 

95 

96 

102 

100 

104 

96 

97 

102 

102 

101 

94 

87 

93 

99 

103 

97 

98 

95 

99 

a Number of animals suMving/number initially in group. Subsequent calculations are based on animals suIviving to the end of the 

AA Significantly different (PSO.01) from the vehicle control group by Dunnett's test 

A Significantly different (PSO.05) from the vehicle control group by Dunnett's test 

*; Significantly different (PSO.01) from the vehicle control group by life table pairwise comparison 

* Significantly different (PSO.05) from the vehicle control group by life table pairwise comparison 

Weights are given as mean f standard error. 

study. 

e Vehicle control group for TPA restart 
Results 59 

remaining 40 weeks (Table 20). In male and female 

and as weekly averages at 10-week intervals for the 

weekly averages for the first 10 weeks of the study 

Incidences of irritation and ulcer are presented as 

localized skin surface excavation and/or broken skin. 

inflamed or appearing sore. Ulcer was defined as 

ulcers. Irritation was defined as the skin being 

The primary clinical findings were irritation and 

1,000 pug MNNG. 

at initiating concentrations of 500 and 

and SENCAR mice exhibited a dose-related increase 

of irritation in all strains and ulcer in Swiss (CD-l@) 

tion never developed ulcers. The percent incidence 

Swiss (CD-l@) mice receiving 100 pg MNNG initiano ulcers were present after week 5. Female 

Swiss (CD-l@) males were observed with ulcers, but 

weeks 2 and 4, 7% of the 100 pg MNNG 

then decreased to zero by weeks 7 to 8. Between 

oped irritation that peaked around weeks 2 to 3 and 

100 pg MNNG initiation followed by acetone develSome male and female Swiss (CD-l@) mice receiving 

zero or near zero for the duration of the study. 

initiation, the incidences of irritation and ulcer were 

D B6C3Fl and SENCAR mice receiving 100 pg MNNG 

trols. 

significantly greater than those of the vehicle conof all three strains receiving 1,000 pg MNNG were 

SENCAR mice receiving 500 pg MNNG and in mice 

The incidences of skin tumors in Swiss (CD-l@) and 

acetone were similar to those of the vehicle controls. 

B6C3F1 mice receiving 500 pg MNNG followed by 

group receiving 100 pg MNNG or in male and female 

(Table 21). The incidences of skin tumors in any 

tion revealed no tumors in the vehicle controls 

Microscopic evaluation of skin at the site of applicaTumor Response in Protocol Controls 

of the vehicle controls (Table 21). 

repeated applications of TPA were greater than those 

male and female Swiss (CD-l@) mice receiving 

controls. However, the incidences of skin tumors in 

applications of TPA were similar to those of vehicle 

B6C3F1 and SENCAR mice receiving repeated 

The incidences of skin tumors in maie and female 

similar to those of the respective vehicle controls. 

mice receiving repeated applications of BPO were 

In all three strains, the incidences of skin tumors in 
60 Skin Paint Studies, NTP TR 441 

Protocol Controls for Study Design B 

and SENCAR Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3Fl Mice, Swiss (CD-l@) Mice, 

TABLE 20 

~ ~- ~~~ 

Week of Study 1 2 3 4 5 6 7 8 9 10 11-20 21-30 31-40 41-50 

20 mg BPODO mg BPO 

5 pg TPN5 pg TPA 

1,OOO pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

B6C3Fl 

Irritation 

Male 

20 mg BPODO mg BPO 

5 pg TPN5 fig TPA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

Swiss (CD-l@) 

20 mg BPODO mg BPO 

1 pg "PAD kg PA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

SENCAR 

0000000000 0 0 0 1 

0030000000 0 0 0 2 

003320 0 0 0 0 0 0 0 0 920 

0000000000 0 0 2 5 

0000000000 0 0 0 2 

347373020 3 3 0 0 0 0 1 0 4 

3 87 100 100 100 87 63 43 43 31 25 20 16 5 

17 97 100 97 97 87 67 57 50 53 36 23 17 3 

3 31 41 69 93 100 66 38 38 43 52 34 20 15 

0 710204327 7 7 7 7 13 13 8 3 

0000000000 0 0 2 1 

027233010 3 3 0 7 3 11 16 13 21 

0 13 20 23 20 20 28 28 34 45 43 34 45 42 

00 0 3 0 7 7232310 12 5 8 7 

0000003333 4 3 0 2 

20 mg BPODO mg BPO 

5 pg TPN5 pg TPA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

B6C3Fl 

Ulcer 

20 mg BPODO mg BPO 

5 pg TPN5 pg TPA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

Swiss (CD-1") 

1,000 pg MNNG/Acetone 

500 fig MNNG/Acetone 

100 pg MNNG/Acetone 

SENCAR 

20 mg BPODO mg BPO 

1 Irg Pg TPA 

0000000000 0 0 0 0 

0000000000 0 0 0 0 

0003000000 0 0 0 0 

0000000000 0 0 0 0 

0000000000 0 0 0 0 

0377000000 0 0 0 1 

0 30 73 67 53 43 37 33 33 31 30 25 5 1 

0 43 87 83 67 47 47 43 40 37 43 27 7 1 

14 21 24 20 

0003333033 5 6 3 1 

0737333334 

0000000000 

0000000000 

020 710 7 3 3 7 7 3 

0000000000 0 0 0 0 

7 14 6 13 

0 60 17 43 47 40 31 34 41 45 39 26 11 10 

23 54 51 31 

2 4 0 0 
Results 61 

Protocol Controls for Study Design B (continued) 

and SENCAR Mice in the Comparative InitiatiodPromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3Fl Mice, Swiss (CD-la) Mice, 

TABLE 20 

Week of Study 1 2 3 4 5 6 7 8 9 10 11-20 21-30 31-40 41-50 

20mgBPOnOmgBPO 7 0 0 0 0 0 0 0 0 0 0 0 3 4 

5 pg TPN5 pg TPA 904310 0 0 0 0 0 0 0 0 0 0 1 

1,000 pg MNNG/Acetone 93 80 47 13 7 3 3 0 0 0 2 2 3 6 

500 pg MNNG/Acetone 70 7 3 0 0 0 0 0 0 0 0 0 0 0 

100 pg MNNG/Acetone 10 0 0 0 0 0 0 0 0 0 0 0 0 0 

B6C3Fl 

Irritation 

Female 

20mg BPOl20 mg BPO 0 7 40 20 20 23 0 0 0 0 0 1 2 3 

5 pg TPN5 pg TPA 7 10 73 87 80 77 67 47 17 20 22 21 19 22 

1,000 pg MNNG/Acetone 40 83 100 100 100 97 90 41 34 34 25 22 11 5 

500 kg MNNG/Acetone 20 93 97 93 97 87 48 24 7 10 5 3 3 0 

100 pg MNNG/Acetone 0 27 43 17 7 3 0 0 0 0 0 0 0 0 

Swiss (CD-la) 

20mg BPOnOmg BPO 0 0 0 0 0 0 0 0 9 3 3 0 1 2 

1 pg TPN1 pg TPA 0 0 7 0 3 10 10 20 23 20 14 9 10 1 

1,000 pg MNNG/Acetone 10 37 7 10 17 10 3 7 17 27 21 16 15 20 

500 pg MNNG/Acetone 0 13 3 0 0 0 0 0 0 0 0 2 5 6 

100 pg MNNG/Acetone 0 0 0 0 0 0 0 0 0 0 0 0 0 1 

SENCAR 

20mg BPOnO mg BPO 0 0 0 0 0 0 0 0 0 0 0 0 2 4 

5 fig TPN5 pg TPA 0000000000 0 0 0 0 

1,000 pg MNNG/Acetone 0 0 3 7 3 3 0 3 3 3 3 3 1 1 

500 pg MNNG/Acetone 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

100 pg MNNG/Acetone 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

B6C3Fl 

Ulcer 

20 mg BPOnO mg BPO 0 0 0 0 0 0 0 0 0 0 0 0 3 4 

5 pg TPN5 pg TPA 0000000000 5 15 13 14 

1,000 pg MNNG/Acetone 13 80 63 67 63 50 45 41 31 31 34 16 1 2 

500 pg MNNG/Acetone 3 57 43 30 17 13 10 10 10 10 4 2 0 0 

100 pg MNNG/Acetone 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Swiss (CD-la) 

20mgBPOnOmgBPO 0 0 0 0 0 0 3 0 0 0 1 0 0 0 

1 pg TPN1 pg TPA 0000000030 25 32 21 13 

1,OOO pg MNNG/Acetone 0 7 3 3 3 3 13 13 17 20 20 10 9 7 

500 pg MNNG/Acetone 0 0 0 0 0 0 0 0 0 0 0 0 1 0 

100 pg MNNG/Acetone 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

SENCAR 
62 Skin Paint Studies, NTP TR 441 

in the Comparative Initiatioflromotion Skin Paint Studies: Protocol Controls for Study Design Ba 

Incidences of Skin Tumors in B6C3FlMice, Swiss (CD-l@) Mice, and SENCAR Mice 

TABLE 21 

B6C3F, Swiss (CD-l@) SENCAR 

Acetone/Acetoneb 

Acetone/Acetone 

Male 

or30 

on0 

22/30** 

19/30** 

2/30 

1,000 kg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

20 mg BPOnO mg BPO 

13/30** 

1/30 

on0 

15/30** 

7/30* * 

1/30 

on0 on0 

4/30' 

- 

- 

2/30 

Acetone/Acetoneb 

Acetone/Acetone 

Female 

OD9 

on0 

1,000 kg MNNG/Acetone 

500 Ig MNNG/Acetone 

100 pg MNNG/Acetone 

20 mg BPOnO mg BPO 

5 crg TPN5 fig TPAd 

1 crg TPN1 pg TPA 

9t30** 

6/29* 

on0 

25t30* * 

8/30* * 

on0 

1/30 

8/28* * - 

a Number of animals with tumors per number of animals with skin examined in-life. Incidences represent clinical observations of 

** PSO.01 

* Significantly different (PjO.05) from the vehicle control group by life table painvise comparison 

SENCAR restart group 

Dose level not administered to this strain 

Vehicle control for TPA restart (SENCAR mice) 

papilloma. 

Because of the high incidences of irritation, ulcer, initiator subthreshold carcinogenic dose. Therefore, 

and skin tumors in the 1,000 pg MNNG initiator groups receiving 1,000 pg MNNG and SENCAR mice 

protocol control mice and the high incidence of skin receiving 500 pg MNNG were excluded from the 

tumors in 500 pg MNNG SENCAR protocol control evaluation of mouse strain skin tumor sensitivity in 

mice, these groups did not meet the criteria for the the initiation/promotion studies. 
Results 63 

MNNG INITIATION 

Survival 

AND TPA PROMOTION 

moribund sacrifice policy. 

removed from the study in accordance with the 

controls. Many of the mice in these groups were 

significantly lower than that of the respective vehicle 

of all mice receiving 1,000 pg MNNGmA was 

their respective vehicle controls (Table 22). Survival 

other 100 pg MNNGmA groups were similar to 

lower than that of the respective vehicle controls; all 

female SENCAR mice receiving 100 pg MNNG was 

Survival of male Swiss (CD-l@) and both male and 

than that of the vehicle controls; final mean body 

receiving 100 pg MNNGfI’PA was significantly lower 

The final mean body weight of female B6C3Fl mice 

Body Weights and Clinical Findings 

(Table 22). 

similar to those of the respective vehicle controls 

weights of all other 100 pg MNNGmA groups were 

(Tables 20 and 23). 

mice receiving TPA alone without initiation 

centage of mice with irritation was similar to that of 

20% of the female Swiss (CD-l@) mice. The perbut still involved more than 38% of the male and 

irritation decreased during the duration of the study 

than in either B6C3Fl or SENCAR mice. This 

higher percentage of male and female Swiss (CD-l@) 

(Table 23). Signs of irritation were observed in a 

for male and female B6C3Fl and SENCAR mice 

betweeiweeks 2 and 3 and fell to zero or near zero 

the percentage of mice with signs of irritation peaked 

surface excavation and/or broken skin. In all strains, 

appearance. Ulcer was defined as localized skin 

ulcer. Irritation was defined as inflamed skin or sore 

The primary clinical findings included irritation and 
64 Skin Paint Studies, NTP TR 441 

in the Comparative InitiatiodPmmotion Skin Paint Studies: MNNG Initiation and TPA Promotion 

Survival and Body Weights of B6C3F, Mice, Swiss (CD-la) Mice, and SENCAR Mice 

TABLE 22 

16/30** 

29/30 

30/30 

30/30 

22/30* * 

29/30 

30/30 

30130 

4/30* * 

lO/30** 

17/30.. 

27/30 

14BO** 

22/30 

23/30 

27/30 

29/30 

lM** 

8/30. * 

9/30' * 

2BO* l 

13t30** 

lorn** 

27/30 

44.1 f 1.lAA 

45.9 f 0.7 

48.1 f 0.6 

47.5 f 0.7 

42.9 f 

44.5 f 0.9A 

45.0 f 0.7A 

47.9 f 1.2 

40.0 f 4.8 

46.1 f 2.3 

48.1 2 1.4 

50.7 k 1.3 

37.2 f 1.0 

40.1 f 1.1 

37.4 f 1.3 

39.0 2 1.1 

38.3' 

51.1 f 2.5 

48.8 f 2.8 

51.7 f 1.4 

42.9 f 2.9 

43.2 1.7 

44.1 f 2.6 

45.7 f 1.7 

93 

97 

101 

90 

93 

94 

79 

91 

95 

95 

103 

96 

74 

99 

94 

94 

94 

97 

a Number of animals surviving/number initially in group. Subsequent calculations arebased on animals surviving to the end of the study. 

AA Significantly different (PSO.01) from the vehicle control group by Dunnett's test 

A Significantly different (PSO.05) from the vehicle control group by Dunnett's test 

** Significantly different (PSO.01) from the vehicle control group by life table painvise comparison 

' No standard error calculated due to high mortality 

Weights are given as mean 2 standard error. 
Results 65 

MNNG Initiation and TPA Promotion 

and SENCAR Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3F, Mice, Swiss (CD-1") Mice, 

TABLE 23 

Week of Study 1 2 3 4 5 6 7 8 9 10 11-20 21-30 31-40 41-50 

1OOpg MNNG/S pg TPA 0 0 40 3 0 3 0 0 0 0 0 0 3 7 

B6C3Fl 

Irritation 

Male 

100 pg MNNG/.5 pg TPA 0 53 90 97 97 93 83 33 53 47 58 42 41 38 

Swiss (CD-1@) 

1OOpg MNNG/S pg TPA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

B6C3Fl 

Ulcer 

100 pg MNNG/l pg TPA 0 0 0 0 0 0 0 0 0 0 15 37 53 43 

SENCAR 

100 pg MNNG/S pg TPA 0 10 43 27 13 10 10 7 7 13 27 42 29 24 

Swiss (CD-l@) 

100 pg MNNG/5 pg TPA 0 30 10 0 0 0 0 0 0 0 0 0 0 1 

B6C3Fl 

Irritation 

Female 

Swiss (CD-1@) 

100 pg MNNG/l pg TF'A 0 0 10 0 0 0 0 0 3 0 7 5 3 0 

SENCAR 

100 pg MNNG/S pg TPA 0 67 90 83 93 73 63 50 30 23 20 23 22 28 

1OOpg MNNGIS pg TPA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

B6C3Fl 

Ulcer 

Swiss (CD-la) 

1OOpg MNNGD pg TPA 0 0 0 0 0 0 0 0 0 0 12 25 21 34 

SENCAR 

100 pg MNNG/.5 pg TPA 0 10 0 0 0 0 0 0 0 0 6 6 21 28 
66 Skin Paint Studies, NTP TR 441 

groups survival was reduced because of the aggressive 

lomas over the course of the 1-year study. In some 

tive percentage of mice that developed skin papilpapillomas. The response is displayed as the cumulaSENCAR mice receiving TPA alone did not develop 

although male and female B6C3Fl and female 

significantly higher than that of the vehicle controls, 

because the tumor response for these groups was 

alone (non-initiated) is included for comparison 

(CD-l@) and male SENCAR mice receiving TPA 

The papilloma response of male and female Swiss 

repeated applications of TPA is shown in Figure 8. 

strains initiated with 100 pg MNNG followed by 

The response of male and female mice of the three 

first tumor, and the number of tumors per mouse. 

tumors, the group mean time to appearance of the 

the number of animals in each group that developed 

measure the skin sensitivity of each mouse strain: 

As in study design A, three parameters were used to 

Tumor Response 

Table 24. 

group that developed skin papillomas is given in 

non-initiated TPA controls. The number of mice per 

SENCAR mice was markedly greater than that of the 

response of male and female Swiss (CD-l@) and male 

removed from the cumulative count. The papilloma 

removed by mechanical grooming. No mice were 

In addition, some tumors may have regressed or been 

moribund sacrifice policy maintained for this study. 

papillomas per mouse. 

SENCAR mice averaged about the same number of 

strains. Male and female Swiss (CD-l@) and 

fewer tumors per mouse than either of the other 

the course of the study. B6C3Fl mice developed 

multiplicity were collected at 10-week intervals over 

sented in Table 25. The data used to calculate tumor 

calculated as in design A and the values are preThe average number of papillomas per mouse was 
Results 67 

la0 100 

,-:-. Swiss CD-1 

'5 

5 Swiss CD-1 (TPA controls) 

0 --e-- SENCAR (TPA controls) Q) 

+ SENCAR 

75 75 

sa 

no. 

Q,€ Ea 

Q,= 22 50 50 

3 

z 

$2 .- 

Y 

I 

Q 

25 25 

0 0 

0 10 20 30 40 50 60 

Week on Test 

-0- SENCAR 

-4- Swiss CD-1 (TPA Controls) 

-4- Swiss 

4 B6C3F1 

Females 

0 

-0- SENCAR 3 

Week on Test 

100 

75 

50 

25 

0 

Followed by 5 Kg [B6C3Fl and Swiss (CD-le)] or 1 kg (SENCAR) TPA Promotion 

Tumor Response of the Three Mouse Strains Initiated with LOO kg MNNG 

FIGURE 8
68 Skin Paint Studies, NTP TR 441 

MNNG Initiation and TPA Promotion 

in the Comparative InitiatiodPromotion Skin Paint Studies: 

Comparisons of Papilloma Response Between B6C3Fl Mice, Swiss (CD-le) Mice, and SENCAR Mice 

TABLE 24 

Acetone 

100 pg MNNG/ 

5 pg TPA 

5 pg TPN 

5 crg TPA 

100 pg MNNG/ 

Life table testd 

Life table test' 

Overall rateb 

Mean weeks to first papilloma' 

Male 

B6C3Fl 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Male 

Swiss (CD-le) 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female 

on0 

e - 

1/30 

15.0 2 0 

4/30 

35.8 k 4.64 

8/30 

34.1 2 4.63 

P=O.O02 

P=O.O02 

9b0 

31.2 * 3.52 

P=O.493 

P=O.493 

1m 

43.0 

P <0.001 

P < 0.001 

16/30 

25.3 f 2.64 

P <0.001 

P < 0.001 

21/30 

21.1 f 2.05 

Acetone 

100 pg MNNG/ 

1 pg TPA 

1 pg TPN 

1 pg TPA 

100 pg MNNG/ 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Male 

SENCAR 

2/30 

47.5 2 4.50 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female - 

on0 

U30 

16.0 2 5.00 

- 

on0 

P<O.OOl 

P <0.001 

16/30 

20.8 f 1.86 

P < 0.001 

P < 0.001 

23/30 

23.0 zk 1.71 

' Mean -C standard deviation 

Beneath the 100 fig MNNG/TPA group incidence is the P value corresponding to pairwise comparison with the TPmA group. 

group. 

Beneath the 100 pg MNNG/TPA group incidence is the P value corresponding to pairwise comparison with the 100 Kg MNNGlAcetone 

Number of animals with tumor per number of animals with skin examined in-life 

e Not applicable; no papillomas obsewed in animal group 
Results 69 

MNNG Initiation and TPA Promotion 

Papillomas Observed During the Comparative InitiationlPromotion Skin Paint Studies: 

TABLE 25 

Week of Study 11 21 31 41 51 

100 pg MNNG/5 pg TPA 

Male 

Swiss (CD-l@) 

100 pg MNNG/5 pg TPA 

Female 

100 pg MNNGfi fig TPA 

Male 

SENCAR 

100 pg MNNGfi pg "A 

Female 

oa ob 00 1.00 0.10 1.00 0.13 1.00 0.21 

00 00 00 00 1.00 0.03 

1.00 0.03 1.50 0.28 00 2.44 1.10 2.00 1.09 

00 1.85 0.83A 00 2.57 1.29A 3.22 1.26A 

00 1.43 0.34 1.75 0.67'. 3.50 1.58 3.75 1.67AT 

00 2.00 0.64A 2.14 1.24A' 1.81 1.53A 2.13 1.31A 

Average number of papillomas expressed as the total number of papillomas/number of mice surviving 

a Average number of papillomas expressed as the total number of papillomas/number of mice with papillomas 

Significantly different (PsO.05) from similarly treated Swiss (CIY-l@) groups by the Mann-Whitney U test 

A Significantly different (PsO.05) from similarly treated B6C3Fl groups by the Mann-Whitney U test 
70 Skin Paint Studies, NTP TR 441 

vehicle controls. 

500 pg MNNG initiation was lower than that of the 

male and female Swiss (CD-1*) mice receiving 

similar to that of the vehicle controls. Survival of 

B6C3Fl mice initiated with 500 pg MNNG was also 

respective vehicle controls (Table 26). Survival of 

initiated with 100 pg MNNG was similar to that of 

Survival of male and female mice of all three strains 

Survival 

AND BPO PROMOTION 

MNNG INITIATION 

controls (Table 26). 

groups were similar to that of their respective vehicle 

final mean body weights of all other MNNGBPO 

significantly lower than that of the vehicle controls; 

initiated with 100, 500, or 1,000 pg MNNG were 

Final mean body weights of female B6C3Fl mice 

Body Weights and Clinical Findings 

served in B6C3Fl mice receiving 500 pg MNNG. 

10 weeks of the study. There were no ulcers ob16% in males and 10% in females during the last 

near zero before increasing again to an average of 

the first 3 weeks, which then decreased to zero or 

increase in the percentage of mice with irritation in 

24% in males or 11% in females. 

group had decreased and did not average greater than 

percentage of animals with signs of irritation in this 

occurring between weeks 3 and 5. By week 10, the 

showed signs of irritation with the peak incidence 

All Swiss (CD-l@) mice receiving 500 pg MNNG 

female Swiss (CD-l*) mice receiving 100 pg MNNG. 

never observed in more than 6% of either male or 

females for the remainder of the study. Ulcer was 

decreased to less than 15% in males and 5% in 

served with irritation peaked at week 3 then 

ulcer. The percentage of 100 pg MNNG mice obdose-related response in the signs of irritation and 

Male and female Swiss (CD-l@) mice exhibited a 

Skin irritation and ulcer were observed in less than The percentage of 500 pg MNNG Swiss (CD-ls) 

10% of male and female B6C3Fl and SENCAR mice mice observed with ulcer also increased during weeks 

receiving 100 pg MNNG initiation (Table 27). 2 and 3, then decreased, and after week 10 did not 

B6C3Fl mice receiving 500 pg MNNG exhibited an exceed 17%. 
Results 71 

in the Comparative InitiatiodPromotion Skin Paint Studies: MNNG Initiation and BPO Promotion 

Survival and Body Weights of B6C3Fl Mice, Swiss (CD-le) Mice, and SENCAR Mice 

TABLE 26 

to Controls (%) 

Relative 

Final Weight 

Initiator/Promoter Survivala Final Mean Body Weightb (9) 

1,000 pg MNNGnO mg BPO 

500 pg MNNGDO mg BPO 

100 pg MNNGDO mg BPO 

20 mg BPOnO mg BPO 

Acetone/Acetone 

Male 

B6C3Fl 

1,000 pg MNNG/20 mg BPO 

500 pg MNNGDO mg BPO 

100 pg MNNGnO mg BPO 

20 mg BPODO mg BPO 

Acetone/Acetone 

Female 

1,000 pg MNNGnO mg BPO 

500 pg MNNGnO mg BPO 

100 pg MNNGDO mg BPO 

20 mg BPOD0 mg BPO 

AcetonelAcetone 

Male 

Swiss (CD-lm) 

1,000 fig MNNGDO mg BPO 

500 pg MNNGDO mg BPO 

100 bg MNNGnO mg BPO 

20 mg BPOnO mg BPO 

AcetonelAcetone 

Female 

1,OOO pg MNNGDO mg BPO 

500 pg MNNGnO mg BPO 

100 pg MNNGnO mg BPO 

20 mg BPOnO mg BPO 

Acetone/Acetone 

Male 

SENCAR 

1,000 pg MNNGDO mg BPO 

500 pg MNNGDO mg BPO 

100 pg MNNGnO mg BPO 

20 mg BPODO mg BPO 

AcetonelAcetone 

Female 

28/30 

29/30 

30/30 

30/30 

30/30 

25/30 

27/30 

30/30 

29/30 

3OBO 

18/30* 

26/30 

2oPo 

27/30 

8/30. l 

17/30** 

m-30. 28/30 

29/30 

27/30 

16/30* * 

28/30 

26Po 

29l30 

6/30* * 

14BO** 

23/30 

29/30 

27/30 

26/30 

46.6 +- 0.8 

46.7 f 0.7 

46.2 f 0.8 

46.8 f 0.8 

47.5 f 0.7 

43.1 k 1.lAA 

43.2 k 1.O.A 

43.8 k 

43.4 f 0.9AA 

47.9 -t 1.2 

47.9 f 2.1 

49.9 f 1.2 

49.4 f 1.2 

48.4 2 1.6 

50.7 f 1.3 

37.6 f 1.2 

36.9 f 1.3 

40.1 f 1.0 

40.7 f 1.1 

39.0 f 1.1 

46.1 k 2.4A 

49.5 * 2.0 

51.2 k 1.2 

52.5 f 1.0 

52.0 f 1.2 

42.7 f 2.0 

46.2 f 1.7 

46.5 k 1.3 

46.2 2 1.8 

44.9 f 1.4 

98 

98 

97 

99 

90 

90 

91 

91 

94 

98 

97 

95 

96 

94 

103 

104 

89 

95 

99 

101 

95 

103 

103 

103 

a Number of animals surviving/number initially in group. Subsequent calculations are based on animals sumiving to the end of the 

AA Significantly different (P50.01) from the vehicle control group by Dunnett’s test 

A Significantly different (P50.05) from the vehicle control group by Dunnett’s test 

** Significantly different (P50.01) from the vehicle control group by life table pairwise comparison 

* Significantly different (PSO.05) from the vehicle control group by life table pairwise comparison 

Weights are given as mean f standard error. 

study. 
72 Skin Paint Studies, NTP TR 441 

MNNG Initiation and BPO Promotion 

and SENCAR Mice in the Comparative Initiation/Promotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3Fl Mice, Swiss (CD-1") Mice, 

TABLE 27 

Week of Study 1 2 3 4 5 6 7 8 9 10 11-20 21-30 31-40 41-50 

5OOpg MNNG/U) mg BPO 0 0 17 0 0 0 0 0 0 0 0 1 2 16 

100pgMNNGnO mg BPO 0 0 0 0 0 0 0 0 0 0 0 0 3 3 

B6C3Fl 

Irritation 

Male 

Swiss (CD-l@) 

lOOpgMNNG/U)mgBPO 0 0 0 0 0 0 0 0 0 3 0 3 5 7 

SENCAR 

500 pg MNNG/U) mg BPO 10 83 100 97 100 80 43 24 17 21 16 19 24 18 

100 pg MNNGD mg BPO 0 47 53 37 30 10 0 3 0 3 8 10 14 3 

500pg MNNGnO mg BPO 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

100pgMNNGTU)mgBPO 0 0 0 0 0 0 0 0 0 0 0 0 2 .3 

B6C3Fl 

Ulcer 

Swiss (CD-l@) 

1OOpg MNNGnO mg BPO 0 0 0 0 0 0 0 0 0 0 0 1 3 2 

SENCAR 

500 pg MNNGnO mg BPO 0 7 33 37 17 20 20 17 14 14 17 16 5 10 

1OOpg MNNG/U) mg BPO 0 0 0 0 0 0 0 0 0 0 0 4 6 4 

(continued\ 
Results 73 

MNNG Initiation and BPO Promotion (continued) 

and SENCAR Mice in the Comparative InitiationPromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as.Percentage) in B6C3F, Mice, Swiss (CD-la) Mice, 

TABLE 27 

~ ~~ ~~ ~~ ~~~~ ~ ~~~ ~~ 

Week of Study 1 2 3 4 5 6 7 8 9 10 11-20 21-30 31-40 41-50 

500 pgMNNG/20 mg BPO 70 3 0 0 0 0 0 0 0 0 0 0 5 10 

1OOpg MNNGnO mg BPO 7 0 0 0 0 0 0 0 0 0 0 0 0 2 

B6C3Fl 

Irritation 

Female 

500 pg MNNGnO mg BPO 37 93 100 97 97 73 30 17 7 10 11 7 4 3 

100 pg MNNG/20 mg BPO 0 43 77 40 0 0 0 3 3 3 4 2 1 0 

Swiss (CD-1@) 

100pgMNNGL2OmgBPO 0 0 0 0 0 0 0 0 3 0 2 0 0 4 

SENCAR 

5OOpg MNNGn0 mg BPO 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

1OOpg MNNGnO mg BPO 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

B6C3F, 

Ulcer 

5OOpg MNNGnO mg BPO 0 47 30 20 13 7 7 7 7 7 13 5 0 0 

1OOpg MNNGnO mg BPO 0 3 3 0 0 0 0 0 0 0 2 0 0 0 

Swiss (CD-l@) 

100pgMNNGnOmgBPO 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

SENCAR 
74 Skin Paint Studies, NTP TR 441 

No mice were removed from the cumulative count. 

regressed or been removed by mechanical grooming. 

this study. In addition, some tumors may have 

aggressive moribund sacrifice policy maintained for 

In some groups survival was reduced because of the 

skin papillomas over the course of the 1-year study. 

the cumulative percentage of mice that developed 

is shown in Figure 9. The response is displayed as 

initiation followed by repeated applications of BPO 

The response of mice receiving 100 pg MNNG 

Tumor Response 

(Table 28). 

100 pg MNNG initiation developed papillomas 

percent of male and female SENCAR mice receiving 

MNNG initiation developed papillomas. Thirty 

three female Swiss (CD-l@) mice receiving 100 pg 

One female B6C3Fl, one male Swiss (CD-la), and 

B6C3Fl and Swiss (CD-l@) mice receiving 500 pg 

Higher incidences of papillomas were observed in 

similar to the respective vehicle controls. 

Swiss (CD-l@) mice receiving 100 pg MNNG were 

B6C3Fl mice receiving 100 or 500 pg MNNG and 

Swiss (CD-l@) mice. The incidences of papilloma in 

groups, but this difference was only significant in 

MNNG than in the vehicle control or 100 pg MNNG 

males and 38.1 weeks for females. 

time to first tumor appearance was 40.6 weeks for 

SENCAR mice receiving 100 pg MNNG, the mean 

18.3 weeks in males and 8.4 weeks in females. In 

times to the appearance of the first papilloma were 

MNNG Swiss (CD-l@) mice, in which the mean 

in females. This time was greatly shortened in 500 pg 

the first papilloma of 38 weeks in males and 31 weeks 

MNNG groups had a mean time to the appearance of 

43 to 47 weeks. In Swiss (CD-l@) mice, the 100 pg 

appearance of the first papilloma in B6C3Fl mice was 

loma is given in Table 28. The mean time to the 

The mean time to the appearance of the first papil- 
Results 75 

'"I 

---+--- Swiss 500 pg 

-+- Swiss 100 pg 

-D- B6C3Fl 500 pg 

75 -+- SENCAR 100 pg 

50 I Males 

100 

75 

a 

0 

.I 

0 10 20 30 

Week on Test 

40 50 

+ SENCAR 100 pg 

--e-- Swiss 500 pg 

-+- Swiss 100 pg 

-0- B6C3F1 500 pg 

-&- B6C3F1 100 pg 

Females 

i 

d 

. 

0: 

A 

8 I 

10 I 

20 1 

30 I 

40 50 

Week on Test 

Followed by 20 mg BPQ Promotion 

Tumor Response of the Three Mouse Strains Initiated with 100 sg MNNG 

FIGURE 9

100 

75 

50 

25 

0 

100 

75 

50 

25 

0 
76 Skin Paint Studies, NTP TR 441 

MNNG Initiation and BPO Promotion 

in the Comparative Initiatioflromotion Skin Paint Studies: 

Comparisons of Papilloma Response Between B6C3Fl Mice, Swiss (CD-1Q) Mice, and SENCAR Mice 

TABLE 28 

100 pg MNNG/ 500 pg MNNG/ 20 mg BPOi 100 pg MNNG/ 500 pg MNNG/ 

Acetone Acetone 20 mg BPO 20 mg BPO 20 mg BPO 

Life table testd 

Life table test' 

Overall rateb 

Mean weeks to first papillomaa 

Male 

B6C3F1 

e - 

1/30 

34.0 - - 

P=O.118 

P=O.308 

3/30 

47.0 2 2.08 

on0 

P=O.O28 

on0 opo 

- 

- 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female - - - 44.0 43.0 & 5.20 

OD0 1/30 3/30 

P=O.500 P=O.114 

P=O.O74 P=O.500 P=O.116 

0/30 on0 

Life table test 

Life table test 

Overall rate 1/30 7/30 

Mean weeks to first papilloma 15.0 * 0 14.9 2 1.60 

Male 

Swiss (CD-l@) 

- 38.0 18.3 k 4.54 

on0 1/30 11/30 

P=O.752 P=O.164 

P <0.001 P=O.522 P < 0.001 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female - 

6/30 

18.0 6.93- 31.0 k 7.57 8.4 * 0.65 

on0 3/30 13/30 

P=O.120 P=O.O32 

P<O.OOl P=O.116 PCO.001 

0/30 

Life table test 

Life table test 

Overall rate 2/30 19/30 

Mean weeks to first papilloma 47.5 k 4.50 13.9 2 2.39 

Male 

SENCAR 

- 40.6 k 2.66 12.9 k 1.91 

on0 9/30 25/30 

P=O.O21 P =0.079 

P<O.OOl P=O.O03 P <0.001 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female - 25.5 * 6.40 47.0 k 0 38.1 & 2.64 25.8 k 4.43 

8/30 1/30 9/30 16/30 

P =0.002 P=O.O46 

PCO.001 P=O.O08 P<O.OOl 

on0 

a Mean 2 standard deviation 

20 mg BPOnO mg BPO group and those groups. 

and 500 pg MNNGnO mg BPO group incidences are the P values corresponding to pailwise comparisons between the 

In the 20 mg BPOnO mg BPO columns are the P values associated with the trend test. Beneath the 100 pg MNNGDO mg BPO 

pailwise comparisons between the appropriate MNNG/Acetone group and those groups. 

Beneath the 100 pg MNNGnO mg BPO and 500 pg MNNGnO mg BPO group incidences are the P values corresponding to 

Number of animals with tumor per number of animals with skin examined in-life 

Value of statistic cannot be computed 

e Not applicable; no papillomas observed in animal group 
Results 77 

The average numbers of papillomas per mouse, cal- there was only one papilloma per mouse except for 

culated as for the MNNG/TYA groups, are presented female SENCAR mice which averaged 1.17 papilin Table 29. Of the mice that developed tumors, lomas at week 41 and 2.00 papillomas at week 52. 

MNNG Initiation and BPO Promotion 

Papillomas Observed During the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE 29 

Week of Study 11 21 31 41 51 

500 pg MNNGDO mg BPO 

100 pg MNNGBO mg BPO 

Male 

B6C3Fl 

500 pg MNNGDO mg BPO 

100 pg MNNGDO mg BPO 

Female 

500 pg MNNGnO mg BPO 

100 pg MNNGDO mg BPO 

Male 

Swiss (CD-le) 

500 pg MNNGnO mg BPO 

100 pg MNNGnO mg BPO 

Female 

100 pg MNNGDO mg BPO 

Male 

SENCAR 

100 pg MNNGnO mg BPO 

Female 

oa ob 

00 00 

00 

00 

00 

00 

00 

1.00 0.07 

00 

00 00 

00 00 00 

00 

00 

00 

00 

1.00 0.03 

00 00 00 1.00 0.04 1.00 0.04 

1.40 0.24A 1.83 0.39A 0 0.08 00 1.00 0.11 

00 1.00 0.03 00 1.00 0.04 1.00 0.04 

1.09 0.47A 1.15 0.47A 0 0.24A 1.00 0.17A 1.00 0.19 

00 00 1.00 0.07 1.00 0.07 1.17 0.39A 

00 1.00 0.03 00 1.17 0.24Ar 2.00 0.55Ar 

Average number of papillomas expressed as the total number of papillomas/number of mice surviving 

a Average number of papillomas expressed as the total number of papillomas/number of mice with papillomas 

Significantly different (PsO.05) from similarly treated Swiss (CD-l@) groups by the Mann-Whitney U test 

A Significantly different (P<O.OS) from similarly treated B6C3Fl groups by the Mann-Whitney U test 
78 Skin Paint Studies, NTP TR 441 

COMPARISON OF MOUSE STRAIN 

trations of TPA (5 pg) than did the SENCAR mice 

though Swiss (CD-l@) mice received higher concen- 

(CD-l@) and SENCAR mice were similar, even 

(Table 30). The incidences of papilloma in Swiss 

significantly greater than those in B6C3Fl mice 

female Swiss (CD-l@) and SENCAR mice were 

Figure 8. The incidences of papilloma in male and 

TPA promotion over the 1-year study is shown in 

(SENCAR) or 5 pg [B6C3Fl and Swiss (CD-l@)] 

mice to 100 pg MNNG initiation and 1 pg 

MNNGITPA: The response of the three strains of 

AS A PROMOTER 

AS AN INITIATOR WITH TPA OR BPO 

RESPONSE TO MNNG 

(1 PcLg). 

response of B6C3Fl mice receiving 100 pg MNNG 

mice are summarized in Table 31. The papilloma 

The in-life papilloma observations for MNNG/TPA 

first papilloma, and average numbers of papillomas. 

incidences, group mean time to the appearance of the 

(CD-l@) and SENCAR mice had similar papilloma 

was lower than either of the other strains. Swiss 

tumor was longer, and the average number of tumors 

lomas, the mean time to the appearance of the first 

mice in all parameters: fewer mice developed papilwas less than that of Swiss (CD-l@) and SENCAR 

similar to that of the vehicle controls (Table 28). 

(CD-1”) mice that received 100 pg MNNG was 

response for male and female B6C3Fl and Swiss 

Swiss (CD-l@) mice (Table 32). The papilloma 

than those in male and female B6C3Fl and male 

loma in SENCAR .mice were significantly greater 

combination of chemicals. The incidences of papilSENCAR mice were the most responsive to this 

tion over the 1-year study is shown in Figure 9. 

mice to 100 pg MNNG initiation and BPO promoMNNGIBPO: The response of the three strains of 

MNNG Initiation and TPA Promotion 

in the Comparative Initiatioflromotion Skin Paint Studies: 

Comparisons of Papilloma Response Between B6C3Fl Mice, Swiss (CD-le) Mice, and SENCAR Mice 

TABLE 30 

B6C3Fl Swiss (CD-le) SENCAR 

Male 

Life table testd 

Life table test’ 

overall b 9/30 

Mean time to first papilloma (weeks) 37.2 

loo pg MNNGA’PA~ 

Life table test 

Life table test 

Overall rate 1/30 

Mean time to first papilloma (weeks) 43.0 

100 pg MNNGA’PA 

Female 

P=O.O02 

16/30 

25.3 

PCO.001 

21/30 

21.1 

P =0.234 

P<O.OOl 

16/30 

20.8 

P=O.296 

P<O.OOl 

23/30 

23.0 

a B6C3F1 mice and Swiss (CD-le) mice received 5 pg TF’A; SENCAR mice received 1 pg TPA. 

P values correspond to pairwise comparison with Swiss (CD-le) mice. 

P values correspond to pairwise comparison with B6C3Fl mice. 

Number of animals with tumor per number of animals with skin examined in-life 
Results 79 

Overview of MNNG/TPA Sensitivity in the Comparative Initiation/Promotion Skin Paint Studies 

TABLE 31 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

with Tumors (Week) Total Micea Mice with Tumorsb 

100 pg MNNG/S pg PA 

Male 

B6C3F, 

100 pg MNNG/S pg TPA 

Female 

9/30 

1/30 

100 pg MNNG/S pg PA 

Male 

Swiss (CD-l@) 

100 pg MNNG/S pg TPA 

Female 

16/30 

21/30 

100 pg MNNW crg 

Male 

SENCAR 

100 pg MNNG/l crg TPA 

Female 

16/30 

23/30 

31.2 

43.0 

25.3 

21.1 

20.8 

23.0 

0.30 

0.03 

1.40 

2.21 

1.47 

2.13 

1 .oo 

1 .oo 

2.63 

3.24 

2.75 

2.78 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

in each group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors 
80 Skin Paint Studies, NTP TR 441 

MNNG Initiation and BPO Promotion 

in the Comparative InitiatiodPromotion Skin Paint Studies: 

Comparisons of Papilloma Response Between B6C3Fl Mice, Swiss (CD-la) Mice, and SENCAR Mice 

TABLE 32 

B6C3Fl Swiss (CD-l@) SENCAR 

Life table test' 

Life table testb 

overall ratea 

Mean weeks to first papilloma 

100 pg MNNGrZO mg BPO 

Male 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

100 pg MNNGD mg BPO 

Female 

1/30 

44.0 

P=O.493 

In0 

38.0 

P=O.284 

3/30 

31.0 

P=0.011 

P=O.O02 

9/30 

40.6 

P~O.062 

P=O.O07 

9/30 

38.1 

a Number of animals with tumor per number of animals with skin examined in-life 

Not applicable; no papillomas in animal group 

P values correspond to pailwise comparison with Swiss (CD-l@) mice. 

P values correspond to pairwise comparison with B6C3Fl mice. 
Results 81 

did the other two strains. Swiss (CD-l@) female 

greater average number of tumors per mouse than 

higher papilloma incidence, SENCAR mice 5 had a 

mice are summarized in Table 33. In addition to a 

The in-life papilloma observations for MNNGBPO 

was similar. 

first tumor in Swiss (CD-l@) and SENCAR males 

papillomas and the mean time to appearance of the 

the first tumor, but only three mice developed 

mice had a shorter mean time to the appearance of 

Overview of MNNG/BPO Sensitivity in the Comparative Initiatioflromotion Skin Paint Studies 

TABLE 33 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

with Tumors Total Micea Mice with Tumorsb (Weeks) 

100 kg MNNGDO mg BPO 

Male 

B6C3F, 

100 kg MNNGnO mg BPO 

Female 

OB0 - C - - 

In0 44.0 0.03 1.00 

100 Kg MNNGnO mg BPO 

Male 

Swiss (CD-la) 

100 kg MNNGDO mg BPO 

Female 

1/30 38.0 0.03 1 .oo 

3/30 31.0 0.13 1.33 

100 kg MNNGDO mg BPO 

Male 

SENCAR 

100 Kg MNNGBO mg BPO 

Female 

9/30 40.6 0.50 1.67 

9/30 38.1 0.60 2.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group 

Not applicable; no papillomas in animal group 

in each group 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors 
82 Skin Paint Studies, NTP TR 441 

100 pg MNNG for the duration of the 1-year study. 

dosed once a week with either 2.5 pg DMBA or 

of male and female mice of the three strains were 

DMBA and MNNG as complete carcinogens, groups 

To compare the mouse strain skin tumor response to 

AS COMPLETE CARCINOGENS 

DMBA AND MNNG 

the study. 

ulcers was never greater than 8% in any strain during 

weeks 41 through 50. The percentage of mice with 

SENCAR mice, which averaged less than 20% during 

male and female Swiss (CD-l@) mice and male 

which averaged 18% during weeks 31 through 40, and 

condition except for female Swiss (CD-l@) mice, 

than 10% of the animals were observed with this 

with signs of irritation was minimal (Table 34). Less 

In mice receiving DMBA, the percentage of mice 

of Application 

Clinical Observations of Skin at the Site 

weeks 31 through 40 and increased to an average of 

averaged 20% in males and 23% in females during 

with skin irritation. However, skin irritation 

B6C3Fl mice (3% during one week) were observed 

first 30 weeks of the study, a very low percentage of 

were observed in MNNG groups (Table 35). In the 

A greater percentage of mice with irritation and ulcer 

was 19% in males and 15% in females. 

weeks 41 through 50 during which time the average 

through 5, and remained low (less than 17%) until 

observed in the Swiss (CD-la) mice during weeks 2 

females during weeks 41 through 50. Ulcers were 

before increasing to 40% in males and 24% in 

percentage generally decreased through week 30 

observed with irritation at weeks 3 to 4. The average 

approximately 90% of males and females were 

observed in Swiss (CD-l@) mice, in which 

percentage of mice with signs of irritation was 

and to 20% during weeks 41 through 50. The highest 

10% in SENCAR males during weeks 31 through 40 

study. The average incidence of ulcers increased to 

and never exceeded 7% for females during the entire 

near zero for most of the first 30 weeks of the study, 

50. The percentage of SENCAR mice with ulcer was 

of 56% males and 64% females by weeks 41 through 

mice with irritation gradually increased to an average 

30. The percentage of male and female SENCAR 

remained relatively constant until weeks 21 through 

females with irritation rose to 7% at week 4 and 

average of 20% in males at week 3; the percentage of 

during the second week of the study, increasing to an 

MNNG than B6C3Fl mice. Irritation appeared 

SENCAR mice were slightly more sensitive to 

incidence averaged 1% in males and 4% in females. 

last 10 weeks of the study during which time the 

50. Ulcers in B6C3Fl mice were not noted before the 

35% in males and females during weeks 41 through 
Results 83 

Repeated Applications of 2.5 kg DMBA 

and SENCAR Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3Fl Mice, Swiss (CD-le) Mice, 

TABLE 34 

~ ~ ~~~~~ 

Week on Study 1-10 11-20 21-30 31-40 41-50 

B6C3F1 

Irritation 

Male 

Swiss (CD-l@) 

SENCAR 

Ulcer 

B6C3Fi 

Swiss (CD-l@) 

SENCAR 

Irritation 

Female 

B6C3F1 

Swiss (CD-la) 

SENCAR 

Ulcer 

B6C3Fl 

SENCAR 

Swiss (CD-le) 

1 

18 

0 

17 

19 

0 

7 

15 

2 

, 
84 Skin Paint Studies, NTP TR 441 

Repeated Applications of 100 pg MNNG 

and SENCAR Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

Incidences of Irritation and Ulcer (Expressed as Percentage) in B6C3Fl Mice, Swiss (CD-l@) Mice, 

TABLE 35 

Week on Study 1 2 3 4 5 6 7 8 9 10 1-20 21-30 31-40 41-50 

B6C3Fj 

Irritation 

Male 

0000300000 0 5 20 35 

Swiss (CD-la) 0 43 83 93 73 62 38 17 21 34 17 15 29 40 

SENCAR 0 17 20 17 13 3 3 10 20 17 14 25 50 56 

Ulcer 

B6C3Fl 0000000000 0 0 0 1 

Swiss (CD-l@) 031713 7 0 0 0 0 0 3 3 7 19 

SENCAR 0000000000 2 1 10 20 

Irritation 

Female 

B6C3Fl 3000000000 0 1 23 35 

Swiss (CD-l@) 0 70 90 83 83 87 50 27 10 13 4 4 17 24 

SENCAR 0337777111 6 18 33 64 

B6C3Fl 

Ulcer 

0000000000 0 0 0 4 

Swiss (CD-l@) 0330000000 0 0 3 15 

SENCAR 0000000003 6 7 6 6 
Results 85 

Swiss (CD-l@) mice. 

tumors was greatest in SENCAR mice and lowest in 

mice. The total number of mice observed with skin 

time to tumor) than in B6C3Fl or Swiss (CD-l@) 

tumors was significantly greater in SENCAR (shorter 

indicated that the response of mice developing 

individual time points over the course of the study 

mice (Table 36). Painvise comparison of strains at 

shortest in SENCAR mice and longest in B6C3Fl 

time to the appearance of the first papilloma was 

tions of DMBA is shown in Figure 10. The mean 

The tumor response in mice receiving topical applica- 

(Table 37). 

significantly greater than that of Swiss (CD-l@) mice 

(CD-l@) mice and that the B6C3Fl response was 

mice than in male B6C3Fl and male and female Swiss 

oping tumors was significantly greater in SENCAR 

the study indicated that the response of mice develstrains at individual time points over the course of 

shorter in SENCAR mice. Pairwise comparison of 

B6C3Fl and Swiss (CD-l@) mice, but was markedly 

time to first appearance of papilloma was similar in 

tions of MNNG is shown in Figure 11. The mean 

The tumor response in mice receiving topical applica- 
86 

25 

0 

Skin Paint Studies, NTP TR 441 

B6C3F1 

-0- SENCAR 

-0- Swiss CD-1 

Males 

I 1 I I I 

10 20 30 40 50 4 

Week on Test 

50 

+ 

100 

75 

50 

25 

0 

Week on Test 

Tumor Response of the Three Mouse Strains Receiving Repeated Applications of 2.5 rg DMBA 

FIGURE 10 
Results 87 

Repeated Applications of 2.5 kg DMBA 

in the Comparative Initiatioflromotion Skin Paint Studies: 

Comparisons of Papilloma Response Between B6C3F, Mice, Swiss (CD-la) Mice, and SENCAR Mice 

TABLE 36 

B6C3Fl Swiss (CD-la) SENCAR 

Life table test' 

Life table testb 

Overall ratea 

Mean weeks to first papilloma 

Male 

25/30 

46.6 (0.86)d 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female 

21/30 

45.3 (0.92) 

P=0.521N 

22/30 

42.9 (1.92) 

P=O.242N 

2OBO 

40.1 (1.97) 

PCO.001 

PCO.001 

21/30 

36.1 (1.36) 

P<O.ool 

P <0.001 

30130 

34.9 (1.49) 

a Number of animals with tumor per number of animals with skin examined in-life 

Mean (standard deviation) 

P values correspond to pairwise comparison with Swiss (CD-la) mice. 

P values correspond to pairwise comparison with B6C3Fl mice. A lower incidence in a dose group is indicated by N. 
88 Skin Paint Studies, NTP TR 441 

100 

75 

50 

25 

0 

Week on Test 

Tumor Response of the Three Mouse Strains Receiving Repeated Applications of 100 crg MNNG 

FIGURE 11 
Results 89 

Repeated Applications of 100 ag MNNG 

in the Comparative Initiatiodl'romotion Skin 'Paint Studies: 

Comparisons of Papilloma Response Between B6C3Fl Mice, Swiss (CD-le) Mice, and SENCAR Mice 

TABLE 37 

B6C3F, Swiss (CD-1@) SENCAR 

Life table test' 

Life table testb 

overall ratea 

Mean weeks to first papilloma 

Male 

27/30 

40.1 (1.40)d 

Life table test 

Life table test 

Overall rate 

Mean weeks to first papilloma 

Female 

37.9 (1.21) 

30/30 

P=0.017N 

17/30 

40.2 (1.93) 

P<0.001N 

19/30 

38.7 (1.92) 

PCO.001 

PcO.001 

29/30 

29.4 (1.50) 

P<O.OOl 

P=O.l5ON 

23/30 

32.3 (1.80) 

' P values correspond to pairwise comparison with Swiss (CD-le) mice. 

a Number of animals with tumor per number of animals with skin examined in-life 

Mean (standard deviation) 

P values correspond to pairwise comparison with B6C3Fi mice. A lower incidence in a dose group is indicated by N. 

somewhat shorter in B6C3Fi mice treated with 

mean time to the appearance of the first tumor was 

with the initiation and promotion treatment. The 

with the complete carcinogen treatment than they did 

of tumors and developed more tumors per animal 

and 39). B6C3Fl mice had a much higher incidence 

as an initiator followed by TPA promotion (Tables 38 

response to the same concentration of the carcinogen 

carcinogen treatment is shown together with their 

The overall sensitivity of mice to the complete 

MNNG complete carcinogen treatment. 

ment than in- those receiving either DMBA or 

greater in mice receiving initiation/promotion treataverage number of tumors per animal was generally 

appearance of the first tumor was shorter and the 

mice were less clear; however, the mean time to the 

papilloma incidence in Swiss (CD-l@) and SENCAR 

complete carcinogen treatment. Differences in the 

DMBA/TPA than it was in mice receiving the DMBA 
90 Skin Paint Studies, NTP TR 441 

Overview of DMBA Sensitivity in the Comparative InitiatioWromotion Skin Paint Studies 

TABLE 38 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

with Tumors Pew Total Micea Mice with Tumorsb 

2.5 pg DMBAn.5 pg DMBA 

2.5 pg DMBN5 pg TPA 

Male 

B6C3Fl 

25/30 

Kv.30 

46.6 

35.4 

2.13 

0.57 

2.56 

1.70 

2.5 pg DMBAn.5 pg DMBA 

2.5 fig DMBN5 fig TPA 

Female 

2780 

3/30 

45.3 

36.7 

2.17 

0.27 

2.41 

2.67 

2.5 pg DMBAn.5 pg DMBA 

2.5 pg DMBNS pg TPA 

Male 

Swim (CD-lm) 

22/30 

27/30 

42.9 

15.4 

1.30 

4.63 

1.77 

5.15 

2.5 pg DMBAn.5 fig DMBA 

2.5 pg DMBNS kg PA 

Female 

2080 

26/30 

40.1 

18.0 

1.27 

3.77 

1.90 

4.35 

2.5 pg DMBAn.5 fig DMBA 

2.5 pg DMBAn pg 

Male 

SENCAR 

2.5 pg DMBAn.5 fig DMBA 

2.5 pg DMBAn pg 'ITA 

Female 

27/30 

19/30 

36.1 

15.5 

2.53 

3.03 

2.81 

4.79 

2.80 

5.23 

2.80 

5.81 

3OBO 

27BO 

34.9 

15.9 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

in each group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors 
Results 91 

Overview of MNNG Sensitivity in the Comparative InitiationPromotion Skin Paint Studies 

TABLE 39 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

with Tumors weew Total Micea Mice with Tumorsb 

27/30 

9/30 

40.1 

37.2 

1.43 

0.30 

1.59 

1.00 

100 pg MNNGDOO pg MNNG 

100 pg MNNG/5 pg TPA 

Female 

30/30 

1/30 

37.9 

43.0 

1.70 

0.03 

1.70 

1.00 

17/30 

16/30 

40.2 

25.3 

0.97 

1.40 

1.71 

2.63 

100 pg MNNGflOO pg MNNG 

100 pg MNNG/S pg TPA 

Female 

19/30 

21/30 

38.7 

21.1 

0.87 

2.27 

1.37 

3.24 

100 pg MNNGD00 pg MNNG 

100 pg MNNG/l pg TPA 

Male 

SENCAR 

29.4 

20.8 

2.00 

1.47 

2.07 

2.75 

29/30 

16/30 

28/30 

23/30 

32.3 

23.0 

1.77 

2.13 

1.89 

2.78 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

in each group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors 

DMBA or MNNG as complete carcinogens, followed 

highest incidence in all three strains of mice receiving receiving DMBA and MNNG initiation. 

Squamous cell carcinoma was the tumor with the mow cell carcinomas in all three strains of mice 

treatment is summarized in Tables 40 and 41. incidences of squamous cell papillomas than squation in mice receiving the complete carcinogen initiation and promotion treatment there were higher 

Microscopic evaluation of skin at the site of applica- by squamous cell papilloma. In mice receiving 
92 Skin Paint Studies, NTP TR 441 

in the Comparative InitiatiodPromotion Skin Paint Studies 

Incidences of Skin Tumors in B6C3Fl Mice, Swiss (CD-la) Mice, and SENCAR Mice 

TABLE 40 

2.5 ag DMBA/ 2.5 ag DMBN 2.5 ag DMBA/ 

2.5 ag DMBA 5 kg TPA 20 mg BPO 

Squamous cell papilloma 

Squamous cell carcinoma 

Keratoacanthoma 

Basal cell carcinoma 

Melanoma NOS 

Sebaceous gland, adenoma 

Male 

B6C3Fl 

Squamous cell papilloma 

Squamous cell carcinoma 

Basal cell carcinoma 

Basal cell adenoma 

Melanoma NOS 

Sebaceous gland, adenoma 

Female 

Squamous cell papilloma 

Squamous cell carcinoma 

Sarcoma 

Keratoacanthoma 

Basal cell adenoma 

Male 

Swiss (CD-19 

Squamous cell papilloma 

Squamous cell carcinoma 

Keratoacanthoma 

Sebaceous gland, adenoma 

Female 

Squamous cell papilloma 

Squamous cell carcinoma 

Keratoacanthoma 

Male 

SENCAR 

Squamous cell papilloma 

Squamous cell carcinoma 

Keratoacanthoma 

Basal cell carcinoma 

Female 

12 

24 

5 

1 

1 

2 

30 

11 

24 

1 

1 

0 

9 

30 

8 

23 

0 

1 

1 

30 

8 

23 

1 

0 

30 

7 

27 

3 

30 

9 

29 

2 

1 

30 

8 

1 

0 

0 

1 

1 

30 

3 

0 

0 

0 

2 

2 

30 

17 

10 

1 

1 

0 

30 

15 

12 

2 

1 

30 

9 

9 

0 

30 

11 

14 

2 

1 

30 

0 

0 

0 

0 

0 

1 

30 

3 

2 

0 

0 

1 

0 

30 

3 

0 

0 

0 

0 

30 

5 

0 

0 

0 

30 

12 

11 

2 

30 

11 

14 

5 

0 

30 
Results 93 

in the Comparative Initiation/Promotion Skin Paint Studies 

Incidences of Skin Tumors in B6C3Fl Mice, Swiss (CD-lm) Mice, and SENCAR Mice 

TABLE 41 

100 pg MNNG/ 100 kg MNNG/ 100 pg MNNG/ 

100 pg MNNG 5 pg TPA 20 mg BPO 

Subcutaneous tissue, squamous cell carcinoma 

Subcutaneous tissue, sarcoma 

Squamous cell papilloma 

Squamous cell carcinoma 

Male 

B6C3Fl 

Subcutaneous tissue, sarcoma 

Subcutaneous tissue, mast cell tumor benign 

Squamous cell papilloma 

Squamous cell carcinoma 

Female 

Squamous cell papilloma 

Squamous cell carcinoma 

Squamous cell carcinoma, metastatic, skin 

Male 

Swiss (CD-P) 

Squamous cell papilloma 

Squamous cell carcinoma 

Keratoacanthoma 

Female 

1 

0 

2 

29 

30 

1 

1 

1 

30 

30 

6 

14 

1 

30 

9 

15 

0 

30 

0 

1 

7 

0 

30 

0 

0 

1 

0 

29 

9 

1 

0 

30 

13 

6 

1 

30 

0 

0 

0 

0 

30 

0 

0 

1 

0 

30 

1 

0 

0 

30 

2 

0 

1 

30 

100 kg MNNG/ 100 pg MNNG/ 100 kg MNNG/ 

100 pg MNNG 1 pg TPA 20 mg BPO 

Squamous cell papilloma 

Squamous cell carcinoma 

Keratoacanthoma 

Basal cell adenoma 

Male 

SENCAR 

Squamous cell papilloma 

Squamous cell carcinoma 

Keratoacanthoma 

Basal cell adenoma 

Female 

7 

27 

0 

1 

30 

1 

1 

30 

7 

28 

4 

5 

0 

0 

30 

10 

6 

0 

0 

30 

8 

2 

2 

0 

30 

6 

2 

2 

0 

30 
94 Skin Paint Studies, NTP TR 441 

complete carcinogen. 

initiated with MNNG or receiving MNNG as a 

DMBA, nor were these lesions observed in mice 

Swiss (CD-l@) or SENCAR mice initiated with 

of the skin. Melanotic lesions were not observed in 

diagnoses were made upon microscopic examination 

Only two lesions were diagnosed grossly; most 

receiving DMBA initiation and TPA promotion. 

incidences of melanoma were greatest in groups 

in groups receiving TPA with no initiation. The 

receiving DMBA initiation and TPA promotion and 

“pigmentation, melanin” were greatest in groups 

melanin” or as “melanoma, NOS.” The incidences of 

within the dermis diagnosed as “pigmentation, 

numbers of deeply pigmented cells (melanocytes) 

(Table 42). Melanotic lesions occurred as increased 

however, the incidences were greatest in female mice 

B6C3Fl mice in the DMBA study (study design A); 

occurred at the site of application in male and female 

Increased incidences of proliferative melanotic lesions 

ally observed extending between cells. 

however, dendritic cellular processes were occasionobscured because of the dense pigmentation; 

polygonal cells (Plate 5). Cellular detail was largely 

composed of closely packed, heavily pigmented 

surrounding subcutaneous adipose tissue. They were 

adjacent dermal adnexal structures and compressed 

were expansive and displaced but did not invade 

subcutaneous adipose tissue (Plate 4). These masses 

the dermis (Plate 3) but frequently extended into the 

multifocal nodular masses that occurred primarily in 

melanocytes. Melanomas were discrete, focal to 

lesions were considered to represent hyperplastic 

distributed within the dermis (Plates 1 and 2). These 

aggregated melanocytes restricted to and randomly 

consisted of focal to multifocal, loosely or densely 

Lesions diagnosed as “pigmentation, melanin” 
Results 95 

Incidences of Melanotic Skin Lesions in B6C3Fl Mice at the Site of Application 

TABLE 42 

Pigmentation, Melanin Melanoma Melanoma, Multiple 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

Acetone/Acetone 

Male 

2.5 pg DMBAIZ.5 pg DMBA 

0n0 

0n0 

MO 

0b0 

1/30 0r30 

2.5 pg DMBAlTPA 

20 mg BPODO mg BPO 

5 pg TPN5 pg TPA 

25 pg DMBMO mg BPO 

2.5 pg DMBAnO mg BPO 

50 pg DMBAATA 

25 pg DMBAATA 

23/30 0b0 

on0 on0 0n0 

14/30 

17/30 

1 on0 

6/30 

2/30 

0/30 

4/30 

2/30 

0n0 

on0 

3/30 

1/30 

on0 

0n0 

0/30 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

AcetoneJAcetone 

Female 

5 pg TPN5 pg TPA 

2.5 pg DMBM.5 pg DMBA 

on0 

on0 

on0 

on0 

7/30 

29/30 0n0 

20 mg BPOD mg BPO 6/30 on0 

50 pg DMBAlTPA 

25 pg DMBAATA 

2.5 pg DMBmA 

11/30 

21/30 

28/30 

9/30 

4/30 

0n0 

25 pg DMBMO mg BPO 

2.5 pg DMBAnO mg BPO 

2/30 

8/30 

4/30 

1/30 
96 Skin Paint Studies, NTP TR 441 
loose aggregates within the papillaly dermis. H&E, 16x 

melanocytes occur as multifocal aggregates in the reticular dermis and as densely and loosely aggregated melanocytes within the dermis. H&E, 4Ox 

receiving 5 (rg TPN5 pg TPA. Increased numbers of deeply pigmented receiving 5 (rg TPN5 (rg TPA. Increased magnification of Plate 1 showing 

Pigmentation, melanin in the skin (site of application) of a B6C3Fl mouse Pigmentation, melanin in the skin (site of application) of a B6C3Fl mouse 

PLATE 1 PLATE 2 
subcutaneous adipose tissue. H&E, 8x 

that is confined largely to the dermis with slight extension into the 

2.5 pg DMBAL2.S pg DMBA Discrete, heavily pigmented, nodular mass 

Melanoma in the skin (site of application) of 'a B6C3Fl mouse receiving 

PLATE 3 

adipose tissue. H&E, 8x carnosus muscle fibers in the subcutis. Cellular detail is obscured by 

nodular mass extends deeply into the subcutis and compresses surrounding composed of heavily pigmented polygonal cells adjacent to the panniculus 

50 Kg DMBN5 Kg TPA Well-circumscribed,heavilypigmented,expansile, 50 pg DMBN5 pg TPA Higher magnification of Plate 4. Melanoma is 

Melanoma in the skin (site of application) of a B6C3Fl mouse receiving Melanoma in the skin (site of application) of a B6C3Fl mouse 

PLATE 4 

extensive accumulation of pigmented granules. H&E, 100~ 

PLATE 5 
97 

DISCUSSION 

tion of that change to the development of a tumor). 

progression from a genetic change through promovaluable information about tumorigenesis (i.e., the 

test agents. Studies of this type have provided 

monitored following treatment of the skin by various 

allows the progress of tumor development to be 

invasive mouse skin initiation/promotion model 

genetic effects (e.g., promoting agents). The nonshort-term tests to detect agents that do not exert 

mendation made that the NTP expand the use of 

These studies were developed to address the recomchemicals. 

standing about the tumorigenic potential of tested 

vide valuable information to increase our undermouse with its large historical database would proObtaining initiation/promotion data in the B6C3Fl 

F344/N rat and B6C3Fl mouse are now well defined. 

reviewed and summarized, and the biology of the 

2-year studies are captured electronically, frequently 

evaluate these chemical effects. Data from NTF' 

and B6C3Fl mouse are the rodent species used to 

study conduct and data collection. The F344/N rat 

studies follow a well-defined standard protocol for 

All of the NTF' 2-year toxicology/carcinogenesis 

ers and complete carcinogens using this protocol. 

the potential of the mouse strains to identify promotand SENCAR mice. The data were used to compare 

skin tumor response data in B6C3Fl, Swiss (CD-la), 

chemicals in an initiation/promotion model to collect 

The current studies used combinations of well-studied 

overall sensitivity to dermal carcinogenicity. 

was intermediate, and B6C3Fl mice showed the least 

criteria, the overall sensitivity of Swiss (CD-l@) mice 

mice relative to the other two strains. By these 

op earlier and to increase in multiplicity in SENCAR 

other two strains. Skin tumors also tended to develrequired to produce effects equivalent to those in the 

lower doses of the test chemical were generally 

appeared to be SENCAR mice, in the sense that 

BPO. The most sensitive of the three strains 

promotion study using DMBA, MNNG, TPA, and 

developing skin tumors in response to the initiation/ 

All three strains of mice demonstrated sensitivity by 

be more reflective of chemical promotion potential. 

Therefore, the skin papilloma response to BPO may 

loma incidence in these two strains (Argyris, 1981). 

TPA may also have contributed to the higher papilulcer in Swiss (CD-l@) and SENCAR mice receiving 

irritating properties of TF'A. The higher incidence of 

mice suggests a difference in skin sensitivity to the 

skin compared with Swiss (CD-l@) and SENCAR 

as a result of TPA application in the B6C3Fl mouse 

papillomas. The low incidence of irritation and ulcer 

than 66% of SENCAR mice were observed with 

strains that received TF'A promotion. However, more 

also developed fewer tumors than did mice of these 

mice receiving DMBA initiation and BPO promotion 

ceived TPA promotion. B6C3Fl and Swiss (CD-l@) 

promotion than for mice of these strains that remice exposed to DMBA initiation followed by BPO 

observations of irritation and ulcer were collected for 

compared with the respective vehicle controls. Fewer 

strains also had significant incidences of papillomas 

incidence of ulcer increased somewhat and these two 

SENCAR mice receiving DMBA/TPA treatment, the 

was significantly increased. In Swiss (CD-la) and 

significantly in B6C3Fl mice, but papilloma incidence 

incidence of irritation and ulcer did not increase 

used as the initiator followed by TF'A promotion, the 

compared with vehicle controls. When DMBA was 

papillomas in the non-initiated Swiss (CD-l@) mice 

5 pg doses of "PA caused an increased incidence of 

ulcer in Swiss (CD-l@) and SENCAR mice. Also, 

any chemical initiation caused skin irritation and 

lesions in any of the strains. However, TPA without 

BPO did not produce significant incidences of these 

irritation or ulcer in non-initiated B6C3Fl mice and 

controls for any of the strains. TPA did not cause 

controls and were rarely observed in DMBA initiator 

irritation or ulcers were observed in acetone vehicle 

hyperplasia (Slaga, 1984). In the current studies, no 

been reported to cause inflammation and epidermal 

studies. Chemicals with promotion potential have 

plastic response to the chemicals used in these 

There were clear differences in mouse skin nonneocarcinoma to squamous cell papilloma in all three 

gens produced a higher ratio of squamous cell 

DMBA and MNNG treatment as complete carcino- 
98 Skin Paint Studies, NTP TR 441 

Reddy and Fialkow, 1983). 

with a carcinogen (DiGiovanni and Juchau, 1980; 

genetic alteration to occur after repeated treatment 

expected given the greater number of times for a 

promoting effect. Also, more carcinomas would be 

of genetic alterations followed by a non-mutagenic 

concept of an initiator producing some fixed number 

promotion treatment. This result would support the 

strains than it did in the mice receiving initiation and 

confined to the dermis. 

1985; Kanno, 1989) and with few exceptions are 

application of chemical carcinogens (Takizawa er al., 

ed in a variety of mouse strains following topical 

attaining a large size. Melanomas have been reportto metastasize to the lungs and other organs upon 

However, spontaneous dermal melanomas are known 

small and considered benign based on morphology. 

B6C3Fl mice in the DMBA studies were relatively 

1989; Bogovski, 1994). The melanomas diagnosed in 

similar to those that occur spontaneously (Kanno, 

melanotic lesions observed in these studies are 

1985) and occur principally in the dermis. The 

B6C3Fl mice (Ward et aZ., 1979; Haseman et d., 

Spontaneous cutaneous melanomas are rare in 

would not be observed in the unpigmented strains. 

not seen in groups receiving MNNG treatment and 

B6C3Fl mice receiving DMBA. These lesions were 

There were increased incidences of melanomas in suspected). 

carcinogenicity studies or effects on melanocytes are 

is a strain-specific response observed in 2-year 

mechanisms of tumor development (e.g., when there 

this strain may, at times, be of more use in explaining 

initiation/promotion protocol, promotion data from 

B6C3Fl mice were less responsive in the skin 

to various initiators and promoters. Though the 

studies, based on the skin tumor response sensitivity 

would be the most acceptable strain to use for such 

mendations to use short-term tests, SENCAR mice 

In response to the NTP ad hoc committee recomin the study of the mechanism of tumorigenesis. 

TG.AC line, has the potential to be extremely useful 

suggest that the use of transgenic mice, such as the 

the short term to tumor, and high tumor yield all 

20 weeks. The absence of the need for an initiator, 

that produced papillomas did so in less than 

been as short as 3 weeks, and all chemicals tested 

some studies, the time to first tumor appearance has 

ment with tumor promoters and carcinogens. In 

epidermal papillomas in response to topical treatinitiated mice, rapidly developing high numbers of 

et al., 1993). TG.AC mice behave like genetically 

ment using the TG.AC transgenic line (Spalding 

begun to study chemical effects on tumor developmodels. For example, NIEHSWTP scientists have 

developed which may replace the existing mouse 

In recent years, transgenic strains of mice have been 

page 8. 

A summary of the Technical Reports Review Subcommittee comments and the public discussion on this Technical Report appears on 
99 

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Hollander, M., and Wolfe, D.A. (1973). 

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Kanno, J. (1989). Melanocytic tumors, skin, mouse. 

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Kaplan, E.L., and Meier, P. (1958). Nonparametric 

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neoplasms for evaluation of rodent carcinogenesis, 

Boorman, G.A. (1986). Guidelines for combining 

McConnell, E.E., Solleveld, HA, Swenberg, J.A., and 

safety assessment. Toxicol. Pafhol. 10, 71-80. 

alterations and hepatocellular tumors in chemical 

Interpretation of rodent hepatocellular proliferative 

Maronpot, R.R., and Boorman, G.A. (1982). 
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181-187. 

experimental blastogenesis. J. Pathol. Bacteriol. 56, 

Mottram, J.C. (1944a). A developing factor in 

391-402. 

experimental blastogenesis. J. Pathol. Bacteriol. 56, 

Mottram, J.C. (1944b). Asensitizing factor in 

Bethesda, MD. 

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U.S. Department of Health, Education, and Welfare, 

Report Series No. 1. NIH Publication No. 76-801. 

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Evaluation, Report. 

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National Toxicology Program (NTP) (1984) Ad Hoc 

carcinogenesis. Nature 322, 78-80. 

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Balmain, A. (1986). Carcinogen-specific mutation 

Quintanilla, M., Brown, K, Ramsden, M., and 

induced by carcinogen alone. Nature 304, 69-71. 

induced by initiation-promotion differ from those 

Reddy, A.L., and Fialkow, P.J. (1983). Papillomas 

365-389. 

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Rous, P., and Kidd, J.G. (1941). Conditional 

Philadelphia, PA. 

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No. 9194M, Benzoyl peroxide IR No. 332, 

nitro-N-nitrosoguanidine IR No. 16655, NMR 

anthracene IR No. 9270, UV No 2413; N-Methy1-N’- 

Sadtler Standard Spectra. 7,12-Dimethylbenz(a)- 

of .Cancer by Vitamins, pp. 10-23. 

specificity of inhibitors. In Modulation and Mediation 

Slaga, T.J. (1983). Multistage skin carcinogenesis and 

Carcinogenesis. CRC Press, Boca Raton, FL. 

Volume ZZ. Tumor Promotion and Skin 

Slaga, T.J. (1984). Mechanisms of Tumor Promotion: 

85-109. 

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differences and solvent effects in mouse skin 

Slaga, T.J., and Fischer, S.M. (1983). Strain 

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Slaga, T.J., Klein-Szanto, A.J.P., Triplett, L.L., and 

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on themechanisms involved in multistage 

Klein-Szanto, A.J.P., and Reiners, J. (1982). Studies 

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Spalding, J.W, Momma, J., Elwell, M.R., and 

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carcinogenesis with 7,12-dimethylbenz(a)anthracene 

melanoma induced in two-stage chemical 

Iwata, IC, and Hayashi, Y. (1985). Mouse skin 

Takizawa, H., Sato, S., Kitajima, H., Konishi, S., 

analysis. Biomenika 62, 679-682. 

Tarone, R.E. (1975). Tests for trend in life table 

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in two-stage carcinogenesis. Prog. Exp. Tumor Res. 

Van Duuren, B.L. (1969). Tumor-promoting agents 

849-854. 

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and Linhart, M.S. (1979). Neoplastic and nonWard, J.M., Goodman, D.G., Squire, R.A., Chu, KC., 

progression. Roc. Soc. Exp. Biol. Med 202, 9-15. 

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Yoshimi, N., Volpe, J.P., Gimenez-Conti, I., and 

Warren, B.S., Naylor, M.F., Winberg, L.D., 
101 

IN THE COMPARATIW INITIATION/PROMOTION 

IN MALE AND FEMALE B6C3Fl MICE 

SUMMARY OF SKIN LESIONS 

APPENDIX A 

SKIN PAINT STUDIES 

TABLE A3d 

TABLE A3c 

TABLE A3b 

TABLE A3a 

TABLE A2d 

TABLE A2c 

TABLE A2b 

TABLE A2a 

TABLE Ald 

TABLE Alc 

TABLE Alb 

TABLE Ala 

in the Comparative Initiation/Promotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female B6C3Fl Mice 

in the Comparative Initiation/Promotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female B6C3Fl Mice 

in the Comparative InitiatiodPromotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male B6C3Fl Mice 

in the Comparative InitiatiodPromotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male B6C3Fl Mice 

Initiatioflromotion Skin Paint Studies: Study Design B 

Overview of the Sensitivity of Female B6C3F, Mice in the Comparative 

InitiationJPromotion Skin Paint Studies: Study Design A 

Overview of the Sensitivity of Female B6C3F, Mice in the Comparative 

InitiatiodPromotion Skin Paint Studies: Study Design B 

Overview of the Sensitivity of Male B6C3F, Mice in the Comparative 

InitiationPromotion Skin Paint Studies: Study Design A 

Overview of the Sensitivity of Male B6C3Fl Mice in the Comparative 

in the Comparative Initiation/Promotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Skin Neoplasms in Female B6C3Fl Mice 

in the Comparative InitiatiodPromotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Skin Neoplasms in Female B6C3Fl Mice 

in the Comparative InitiatiodPromotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Skin Neoplasms in Male B6C3Fl Mice 

in the Comparative InitiatiodPromotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Skin Neoplasms in Male B6C3Fl Mice 

....... 

116 

114 

112 

111 

111 

110 

110 

108 

106 

104 

102 

....... 

....... 

....... 

....................... 

....................... 

....................... 

....................... 

....... 

....... 

....... 

....... 118 
102 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Skin Neoplasms in Male B6C3Fl Mice in the Comparative Initiatioflromotion 

TABLE Ala 

Acetone/ 2.5 pg DMBA/ 2.5 pg DMBA/ 2.5 pg DMBA/ 2.5 pg DMBN 5 pg TPN 

Acetone Acetone 2.5 pg DMBA 5 pg TPA 20 mg BPO 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

30 30 30 30 30 

26 

1 

3 

26 

1 

3 

27 

1 

2 

Suwivors 

30 30 

30 

1 

29 

Animals examined microscopically 30 30 30 30 30 30 

Sebaceous gland, site of 

Melanoma NOS 

Skin 

melanoma NOS, multiple 

Site of application-no mass, 

melanoma NOS 

Site of application-no mass, 

application-mass, adenoma, multiple 

Sebaceous gland, site of 

application-mass, adenoma 

Site of application-mass, 

basal cell carcinoma 

Site of application-mass, 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma, multiple 

Site of application-mass, 

keratoacanthoma 

Subcutaneous tissue, site of 

Subcutaneous tissue, sarcoma 

squamous cell papilloma, multiple 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, fibrosarcoma 

(30) 

1 (3%) 

1 (3%) 

1 (3%) 1 (3%) 1 (3%) 

1 (3%) 

4 (13%) 

14 (47%) 

10 (33%) 

1 (3%) 

7 (23%) 

5 (17%) 

1 (3%) 

1 (3%) 

4 (13%) 

4 (13%) 

1 (3%) 

Neoplasm Summary 

Total animals with uncertain neoplasmsTotal animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasmsb 

benign or malignant 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

Total uncertain neoplasms 

26 

26 

19 

17 

46 

29 

1 

1 

1 

1 

9 

9 

11 

11 

1 

1 

1 

1 

1 

1 
Sldn PIllat Studies: Study Design A (conhued) 

Summary of the Incldcacc of Sldn Neoplasms in Male B6C3FI Mte in the Comparative InitMoalpromotlon 

TABLE Ala 

30 

30 

30 

Neoplasm Summay (continued) 

Total animals with uncertain neoptasmsTotal animals with malignant neoplasms 

Total animala with benign neoplasms 

Total animals with primacy neoplasms 

benign or malignant 

Total malignant neoplasms 

Total benign neoplasms 

Total primacy neoplasm 

Total uncertain neoplasms 

1 

1 

1 

1 

14 

11 

19 

14 

1 

1 

4 

4 

1 

1 

20 

16 

34 

21 

4 

4 

10 

10 

primary neoplasms: all neoplasms except metastatic neoplasms 

Number of animals examined microscopically at site and number of animals with neoplasm 

.. 
104 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Skin Neoplasms in Male B6C3Fl Mice in the Comparative Initiation/Promotion 

TABLE Alb 

Acetone/ 5 pg TPN 100 pg MNNG/ 100 pg MNNGI 100 pg MNNG/ 100 ag MNNGI 

Acetone 5 pg TPA Acetone 100 pg MNNG 20 mg BPO 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

30 30 30 

Survivors 

30 30 30 

30 30 

2 

12 

16 30 

1 

30 

29 

Animals examined microscopically 30 30 30 30 30 30 

Skin 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

application-mass, hemangiosarcoma 

Subcutaneous tissue, site of 

application-mass fibrosarcoma 

Subcutaneous tissue, site of 

application-no mass, sarcoma 

Subcutaneous tissue, site of 

application-no mass, lymphangiosarcoma 

Subcutaneous tissue, site of 

application-no mass, lymphangioma 

Subcutaneous tissue, site of 

trichoepithelioma 

Site of application-mass, 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, squamous cell carcinoma 

Subcutaneous tissue, site of 

application-mass, sarcoma, multiple 

(30) (30) (30) (30) 

1 (3%) 

2 (7%) 

13 (43%) 

16 (53%) 

5 (17%) 

2 (7%) 

1 (3%) 

Neoplasm Summary 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasmsb 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

30 

29 

2 

2 

32 

29 
Lesions in Male and Female B6C3Fl Mice 105 

Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Skin Neoplasms in Male B6C3Fl Mice in the Comparative Initiation/Promotion 

TABLE Alb 

500 pg MNNGI 500 pg MNNGI 1,000 pg MNNCI 1,000 pg MNNGI 1,000 pg MNNGI 20 mg BPOI 

Acetone 20 mg BPO Acetone 20 mg BPO 5 pg TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

30 

1 

30 

2 

30 

1 

SuMvors 

29 29 28 

Animals examined microscopically 30 30 30 

2 

30 30 30 

14 

28 

30 

16 

30 

30 

30 

Skin (continued) 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

(30) (30) (30) 

squamous cell carcinoma 

keratoacanthoma 

2 (7%) 3 (10%) 

1 (3%) 

application-mass, fibrosarcoma 

application-no mass, sarcoma 

application-no mass, lymphangiosarcoma 

application-no mass, lymphangioma 

trichoepithelioma 

squamous cell papilloma, multiple 

squamous cell papilloma 

squamous cell carcinoma, multiple 

Subcutaneous tissue, site of 

Subcutaneous tissue, site of 

Subcutaneous tissue, site of 

Subcutaneous tissue, site of 

Subcutaneous tissue, site of 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

1 (3%) 

1 (3%) 

1 (3%) 

application-mass, hemangiosarcoma 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, squamous cell carcinoma 

Subcutaneous tissue, site of 

application-mass, sarcoma, multiple 

1 (3%) 1 (3%) 3 (10%) 

1 (3%) 

3 (10%) 3 (10%) 

1 (3%) 1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

2 (7%) 

3 (10%) 

4 (13%) 

1 (3%) 

3 (10%) 10 (33%) 

1 (3%) 2 (7%) 

1 

Total animals with malignant neoplasms 1 

Total animals with benign neoplasms 

1 

Total animals with primary neoplasms 1 

Neoplasm Summary (continued) 

4 

4 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

4 

4 

8 

8 

2 

2 

10 

10 

6 

6 

4 

4 

10 

9 

18 

16 

9 

8 

27 

22 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 
106 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Skin Neoplasms in Female B6C3Fl Mice in the Comparative Initiationpromotion 

TABLE Alc 

Acetone Acetone 2.5 pg DMBA 5 pg TPA 20 mg BPO 5 pg TPA 

Acetonel 2.5 DMBN 2.5 kg DMBN 2.5 pg DMBN 2.5 kg DMBN 5 pg TPN 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

30 30 

29 

1 

SuMvors 

30 

30 30 30 

2 

2 

1 

1 

26 30 28 

30 

30 

Animals examined microscopically 30 30 30 30 30 30 

Sebaceous gland, site of 

Squamous cell carcinoma 

Skin 

Sebaceous gland, site of 

(30) (30) (30) (30) (30) (30) 

application-mass, adenoma 8 (27%) 2 (7%) 

melanoma NOS, multiple 

Site of application-no mass, 

melanoma NOS 

Site of application-no mass, 

application-mass, adenoma, multiple 

Site of application-mass, 

basal cell carcinoma 

Site of application-mass, 

basal cell adenoma 

Site of application-mass, 

melanoma NOS 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

application-mass, hemangiosarcoma 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

1 (3%) 

2 (7%) 1 (3%) 

8 (27%) 

16 (53%) 

1 (3%) 

1 (3%) 

2 (7%) 

2 (7%) 

3 (10%) 1 (3%) 3 (10%) 

8 (27%) 

1 (3%) 

Neoplasm Summary 

Total animals with uncertain neoplasmsTotal animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primaly neoplasmsb 

benign or malignant 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

Total uncertain neoplasms 

25 

25 

21 

14 

46 

29 

2 

2 

1 

1 

1 

1 

1 

1 
Lesions in Male and Female B6C3Fl Mice 

Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Skin Neoplasms in Female B6C3F, Mice in the Comparative Initiatioflromotion 

TABLE Alc 

107 

20 mg BPO 

25 pg DMBA/ 25 pg DMBA/ 25 pg DMBN 50 pg DMBA/ 50 pg DMBA/ 20 mg BPO/ 

Acetone 5 pg TPA 20 mg BPO Acetone 5 Irg PA 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

30 30 30 30 30 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 28 

1 

1 1 

27 

1 

2 

Survivors 

30 29 30 

Animals examined microscopically 30 30 30 30 30 

2 

1 

30 

30 

27 

Sebaceous gland, site of 

Squamous cell carcinoma 

Skin (continued) 

melanoma NOS, multiple 

Site of application-no mass, 

melanoma NOS 

Site of application-no mass, 

application-mass, adenoma, multiple 

Sebaceous gland, site of 

application-mass, adenoma 

Site of application-mass, 

basal cell carcinoma 

Site of application-mass, 

basal cell adenoma 

Site of application-mass, 

melanoma NOS 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

application-mass, hemangiosarcoma 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

(30) (30) (30) (30) (30) (30) 

3 (10%) 

1 (3%) 

1 (3%) 

1 (3%) 

9 (30%) 

10 (33%) 

2 (7%) 

11 (37%) 

4 (13%) 

5 (17%) 4 (13%) 

3 (10%) 

1 (3%) 

3 (10%) 1 (3%) 

1 (3%) 

1 (3%) 

5 (17%) 

5 (17%) 

~~ 

Neoplasm Summary (continued) 

Total animals with uncertain neoplasmsTotal animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasms 

benign or malignant 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

Total uncertain neoplasms 

~ ~~ 

4 

3 

7 

7 

20 

13 

9 

9 

24 

17 

46 

24 

20 

19 

2 

2 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 
108 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Skin Neoplasms in Female B6C3Fl Mice in the Comparative Initiatioflromotion 

TABLE Ald 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

30 30 30 

Animals examined microscopically 30 

Missing 

survivors 

30 30 30 

30 30 

30 30 

4 

13 

13 

1 

29 

30 29 

30 

30 

30 

Sarcoma 

skin 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

application-mass, adenoma 

Sebaceous gland, site of 

Squamous cell carcinoma 

Subcutaneous tissue, site of 

Subcutaneous tissue, control, sarcoma 

squamous cell papilloma, multiple 

application-no mass, sarcoma 

Subcutaneous tissue, site of 

application-no mass, lymphangioma 

mast cell tumor benign 

application-no mass, 

Subcutaneous tissue, site of 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, sarcoma, multiple 

1 (3%) 

6 (20%) 

24 (80%) 

1 (3%) 

1 (3%) 

1 (3%) 

Neoplasm Summary 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasms b 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

31 

30 

2 

2 

33 

30 
Lesions in Male and Female B6C3F, Mice 109 

Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Skin Neoplasms in Female B6C3Fl Mice in the Comparative Initiation/Promotion 

TABLE Ald 

100 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 20 mg BPO/ 

20 mg BPO 20 mg BPO Acetone 20 mg BPO 5 pg TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

30 30 30 30 

2 

1 

23 

1 

6 5 

Missing 

Sulvivors 

30 27 25 

Animals examined microscopically 30 30 30 30 

30 30 

3 

5 1 

22 29 

30 30 

Sebaceous gland, site of 

Squamous cell carcinoma 

Sarcoma 

Skin (continued) 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

application-mass, adenoma 

Subcutaneous tissue, site of 

Subcutaneous tissue, control, sarcoma 

squamous cell papilloma, multiple 

application-no mass, sarcoma 

Subcutaneous tissue, site of 

application-no mass, lymphangioma 

mast cell tumor benign 

application-no mass, 

Subcutaneous tissue, site of 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, sarcoma, multiple 

(30) 

1 (3%) 

(30) (30) 

1 (3%) 

1 (3%) 

1 (3%) 

5 (17%) 4 (13%) 1 (3%) 

1 (3%) 

2 (7%) 1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 4 (13%) 3 (10%) 7 (23%) 

1 (3%) 1 (3%) 

Neoplasm Summary (continued) 

1 

Total animals with benign eoplasms 1 

1 

Total animals with primary neoplasms 1 

Total animals with malignant neoplasms 

1 

1 

Total malignant neoplasms 

Total benign neoplasms 

Total primaly neoplasms 

1 

1 

13 

9 

8 

8 

3 

3 

11 

11 

9 

8 

3 

3 

12 

10 

13 

9 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 
110 Skin Paint Studies, NTP TR 441 

Study Design A 

Overview of the Sensitivity of Male B6C3Fl Mice in the Comparative Initiation/Promotion Skin Paint Studies: 

TABLE A2a 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors Total Micea Mice with Tumorsb (Weeks) 

2.5 fig DMBAn.5 pg DMBA 

25 pg DMBAnO mg BPO 

2.5 pg DMBAn0 mg BPO 

50 pg DMBN5 kg TPA 

25 pg DMBN5 pg TPA 

2.5 pg DMBN5 kg TPA 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

20 mg BPODO mg BPO 

5 pg TPN5 pg TPA 

Acetone/Acetone 

25/30 

1/30 

1/30 

22/30 

12/30 

10/30 

OB0 

on0 

0/30 

on0 

OB0 

of30 

- 

- 

46.6 

39.0 

48.0 

32.5 

27.6 

35.4 

- 

2.13 

0.03 

0.03 

1.95 

1.13 

0.57 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

2.56 

1.00 

1.00 

2.66 

2.83 

1.70 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 

Study Design B 

Overview of the Sensitivity of Male B6C3Fl Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE A2b 

With Tumors 

Number of Mice to Tumor 

Mean Time Average Number Average Number 

of Tumors/ of Tumors/ 

Week) Total Micea Mice with Tumorsb 

100 pg MNNG/100 pg MNNG 

1,000 pg MNNG/20 mg BPO 

500 fig MNNGDO mg BPO 

100 pg MNNGDO mg BPO 

1,000 pg MNNG/5 pg TPA 

100 pg MNNG/5 pg TPA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

20 mg BPODO mg BPO 

5 pg TPN5 pg TPA 

Acetone/Acetone 

27/30 

9/30 

3/30 

0/30 

26/30 

9/30 

13/30 

1/30 

OB0 

0/30 

on0 

on0 - 

- 

16.6 

37.2 

23.8 

34.0 

- 

- 

40.1 

34.4 

47.0 

1.43 

0.33 

0.10 

0.00 

1.30 

0.30 

0.43 

0.03 

0.00 

0.00 

0.00 

0.00 

1.59 

1.11 

1 .oo 

0.00 

1.50 

1 .oo 

1.00 

1.00 

0.00 

0.00 

0.00 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 
Lesions in Male and Female B6C3Fl Mice 111 

Study Design A 

Overview of the Sensitivity of Female B6C3Fl Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE A2c 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors Pew Total Micea Mice with Tumorsb 

2.5 pg DMBAn.5. pg DMBA 

2.5 Fg DMBAnO mg BPO 

2.5 bg DMBA/20 mg BPO 

50 pg DMBA/5 pg TPA 

25 pg DMBN5 pg TPA 

2.5 pg DMBNS pg TPA 

50 pg DMBNAcetone 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

20 mg BPOm mg BPO 

5 pg TPN5 pg TPA 

Acetone/Acetone 

45.3 

44.0 

51.0 

40.8 

41.0 

36.7 

- 

2.17 

0.07 

0.13 

1.10 

0.37 

0.27 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

2.41 

1.00 

1 .oo 

2.06 

1.57 

2.67 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 

Study Design B 

Overview of the Sensitivity of Female B6C3Fl Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE A2d 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors Total Micea Mice with Tumorsb (Week) 

100 pg MNNGB00 pg MNNG 

1,OOO pg MNNGIU) mg BPO 

500 pg MNNGIU) mg BPO 

100 pg MNNGIU) mg BPO 

1,000 fig MNNG/5 fig TPA 

100 pg MNNG/5 pg TPA 

1,OOO pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

20 mg BP0m mg BPO 

5 pg TPN5 pg TPA 

AcetonelAcetone 

3ON 

13/30 

3/30 

1no 

16/30 

1/30 

13i3 

OB0 

on0 

or30 

0/30 

on0 

37.9 

24.5 

43.0 

44.0 

25.8 

43.0 

16.0 

1.70 

0.50 

0.10 

0.03 

0.70 

0.03 

0.57 

0.00 

0.00 

0.00 

0.00 

0.00 

1.70 

1.15 

1 .oo 

1.00 

1.31 

1.00 

1.31 

0.00 

0.00 

0.00 

0.00 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 
112 Skin Paint Studies, NTP TR 441 

Initiatioflromotion Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male B6C3Fl Mice in the Comparative 

TABLE A3a 

Acetone/ 2.5 fig DMBN 2.5 pg DMBN 2.5 pg DMBN 2.5 pg DMBN 5 pg TPN 

Acetone Acetone 2.5 pg DMBA 5 pg TPA 20 mg BPO 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

30 30 30 30 30 30 

26 

1 

3 

26 

1 

3 

27 

1 

2 1 

SuMvors 

30 30 29 

Animals examined microscopically 30 30 30 30 30 30 

acanthosis 

Site of application-no mass, 

Skin 

Site of application-no mass, 

cyst epithelial inclusion 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

granuloma 

Site of application-no mass, 

pigmentation, melanin 

Site of application-no mass, 

acanthosis 

Site of application-mass, 

ulcer, multiple 

Site of application-no mass, 

ulcer 

(30) 

1 (3%) 

5 (17%) 28 (93%) 

2 (7%) 

2 (7%) 1 (3%) 

1 (3%) 1 (3%) 2 (7%) 1 (3%) 

10 (33%) 3 (10%) 23 (77%) 

27 (90%) 29 (97%) 
Lesions in Male and Female B6C3Fl Mice 113 

InitiatiodPromotion Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male B6C3Fl Mice in the Comparative 

TABLE A3a 

25 pg DMBN 25 pg DMBN 25 pg DMBN 50 pg DMBN 50 Kg DMBN 20 mg BPO/ 

Acetone 5 kg TPA 20 mg BPO Acetone 5 pg TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

1 

30 30 30 30 

1 

Survivors 

29 29 30 30 

Animals examined microscopically 30 30 30 30 

30 30 

3 

1 

30 

26 

30 

30 

acanthosis 

Site of application-no mass, 

Skin (continued) 

Site of application-no mass, 

cyst epithelial inclusion 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

granuloma 

Site of application-no mass, 

pigmentation, melanin 

Site of application-no mass, 

ulcer, multiple 

Site of application-no mass, 

ulcer 

(30) (30) 

8 (27%) 29 (97%) 

1 (3%) 28 (93%) 

17 (57%) 

Site of application-mass, 

acanthosis 2 (7%) 

(30) (30) (30) (30) 

30 (100%) 7 (23%) 29 (97%) 27 (90%) 

27 (90%) 

2 (7%) 

1 (3%) 

1 (3%) 

28 (93%) 26 (87%) 

14 (47%) 

1 (3%) 

1 (3%) 

~~ ~ ~~ ~~ ~~ ~~~~~ 

a Number of animals examined microscopically at site and number of animals with lesion 
114 Skin Paint Studies, NTP TR 441 

Initiation/Promotion Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male B6C3F, Mice in the Comparative 

TABLE A3b 

Acetone/ 5 pg TPN 100 pg MNNG/ 100 pg MNNG/ 100 pg MNNG/ 100 pg MNNG/ 

Acetone 5 pg TPA Acetone 100 pg MNNG 20 mg BPO 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

30 30 30 

16 

2 

12 

30 

Survivors 

30 30 30 

30 

30 

1 

30 

29 

Animals examined microscopically 30 30 30 30 30 30 

Hair follicle, site of 

Inflammation, chronic active 

Skin 

acanthosis 

Site of application-no mass, 

application-mass, hyperplasia 

Sebaceous gland, site of 

application-no mass, cyst 

(30) (30) (30) (30) 

2 (7%) 2 (7%) 

21 (70%) 22 (73%) 29 (97%) 

application-no mass, granuloma 

Subcutaneous tissue, site of 

hyperplasia, basal cell 

Site of application-mass, 

acanthosis 

Site of application-mass, 

ulcer 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

12 (40%) 

30 (100%) 2 (7%) 

2 (7%) 

15 (50%) 9 (30%) 20 (67%) 

9 (30%) 

1 (3%) 

3 (10%) 1 (3%) 

1 (3%) 
Lesions in Male and Female B6C3Fl Mice 115 

Initiatioflromotion Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male B6C3Fl Mice in the Comparative 

TABLE A3b 

500 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNC/ 20 mg BPO/ 

Acetone 20 mg BPO Acetone 20 mg BPO 5 pg TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 1 

30 

1 

30 

2 

30 

Survivors 

29 29 28 

2 

30 

14 

30 

28 16 

30 

30 

Animals examined microscopically 30 30 30 30 30 30 

Hair follicle, site of 

Inflammation, chronic active 

Skin (continued) 

application-no mass, granuloma 

Subcutaneous tissue, site of 

hyperplasia, basal cell 

Site of application-mass, 

acanthosis 

Site of application-mass, 

ulcer 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-mass, hyperplasia 

Sebaceous gland, site of 

application-no mass, cyst 

(30) 

18 (60%) 

1 (3%) 

(30) (30) (30) (30) (30) 

1 (3%) 

1 (3%) 1 (3%) 

1 (3%) 1 (3%) 

1 (3%) 5 (17%) 

27 (90%) 19 (63%) 25 (83%) 28 (93%) 

4 (13%) 4 (13%) 

9 (30%) 5 (17%) 19 (63%) 20 (67%) 

1 (3%) 

1 (3%) 1 (3%) 1 (3%) 2 (7%) 

1 (3%) 3 (10%) 

12 (40%) 

24 (80%) 

~~ 

a Number of animals examined microscopically at site and number of animals with lesion 
116 Skin Paint Studies, NTP TR 441 

Initiation/Promotion Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female B6C3Fl Mice in the Comparative 

TABLE A3c 

Acetonel 2.5 pg DMBN 2.5 pg DMBN 2.5 pg DMBN 2.5 pg DMBN 5 pg TPN 

Acetone Acetone 2.5 pg DMBA 5 pg TPA 20 mg BPO 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

30 30 30 30 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

SuMvors 

2 

2 

1 

30 29 26 30 

Animals examined microscopically 30 30 30 30 

30 

1 

1 

28 

30 

30 

30 

30 

~~~ ~ ~~~ ~ 

mineralization 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

granuloma 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-mass, hyperplasia 

Sebaceous gland, site of 

Skin 

Site of application-no mass, 

pigmentation, melanin 

Site of application-no mass, 

acanthosis 

Site of application-mass, 

ulcer, multiple 

Site of application-no mass, 

ulcer 

(30) 

1 (3%) 

24 (80%) 

1 (3%) 

9 (30%) 

7 (23%) 

1 (3%) 

2 (7%) 

3 (10%) 

30 (100%) 29 (97%) 30 (100%) 

30 (100%) 29 (97%) 30 (100%) 

28 (93%) 8 (27%) 9 (97%) 

1 (3%) 

1 (3%) 2 (7%) 1 (3%) 

2 (7%) 1 (3%) 
Lesions in Male and Female B6C3Fl Mice 117 

InitiatiodPromotion Skin Paint Studies: Study Desjgn A (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female B6C3Fl Mice in the Comparative 

TABLE A3c 

25 pg DMBN 25 pg DMBN 25 pg DMBA/ 50 pg DMBN 50 pg DMBA/ 20 mg BPO/ 

Acetone 5 pg TPA 20 mg BPO Acetone 5 pg TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

30 30 30 

28 

1 

1 1 

SuMvors 

30 29 

30 30 

1 

2 

30 27 

Animals examined microscopically 30 30 30 30 30 

30 

2 

1 

30 

27 

~ ~~ ~ ~ 

mineralization 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

granuloma 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-mass, hyperplasia 

Sebaceous gland, site of 

Skin (continued) 

Site of application-no mass, 

pigmentation, melanin 

Site of application-no mass, 

acanthosis 

Site of application-mass, 

ulcer, multiple 

Site of application-no mass, 

ulcer 

(30) 

5 (17%) 

1 (3%) 

30 (100%) 28 (93%) 18 (60%) 30 (100%) 29 (97%) 

30 (100%) 30 (100%) 2 (7%) 30 (100%) 27 (90%) 

1 (3%) 

21 (70%) 2 (7%) 

3 (10%) 1 (3%) 

1 (3%) 

11 (37%) 6 (20%) 

3 (10%) 

1 (3%) 

a Number of animals examined microscopically at site and number of animals with lesion 
118 Skin Paint Studies, NTP TR 441 

InitiatiodPromotion Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female B6C3Fl Mice in the Comparative 

TABLE A3d 

Acetonel 5 pg TPA/ 100 pg MNNGI 100 pg MNNGI 100 pg MNNG/ 500 pg MNNG/ 

Acetone 5 p TPA Acetone 100 pg MNNG 5 pg TPA Acetone 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

30 30 30 30 

4 

13 

Missing 

Survivors 

Terminal sacrifice 30 30 30 13 

30 30 

1 

29 30 

Animals examined microscopically 30 30 30 30 29 30 

Sebaceous gland, site of 

Hair follicle, site of 

Skin (30) (30) (30) (30) (29) (30) 

application-mass, hyperplasia 

application-no mass, cyst 1 (3%) 

application-no mass, fibrosis 

Subcutaneous tissue, site of 

acanthosis 

Site of application-mass, 

ulcer 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

cellular, focal, mast cell 

application-no mass, infiltration 

Subcutaneous tissue, site of 

application-no mass, mineralization 

Subcutaneous tissue, site of 

27 (90%) 

30 (100%) 2 (7%) 

12 (40%) 

17 (57%) 29 (100%) 6 (20%) 

25 (83%) 29 (100%) 20 (67%) 

1 (3%) 

1 (3%) 

1 (3%) 
Lesions in Male and Female B6C3Fl Mice 119 

Initiatioflromotion Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female B6C3F, Mice in the Comparative 

TABLE A3d 

100 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 20 mg BPOI 

20 mg BPO 20 mg BPO Acetone 20 rng BPO 5 pg TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

30 

2 

1 

30 30 30 

23 

1 

6 5 

Animals examined microscopically 30 

Missing 

SuMvors 

30 27 25 

30 30 30 

30 30 

3 

5 1 

22 29 

30 30 

application-no mass, fibrosis 

Subcutaneous tissue, site of 

acanthosis 

Site of application-mass, 

ulcer 

Site of application-no mass, 

inflammation, chronic active 17 (57%) 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-mass, hyperplasia 

Sebaceous gland, site of 

application-no mass, cyst 

Hair follicle, site of 

Skin (continued) 

cellular, focal, mast cell 

application-no mass, infiltration 

Subcutaneous tissue, site of 

application-no mass, mineralization 

Subcutaneous tissue, site of 

(30) 

22 (73%) 

(30) (30) (30) 

1 (3%) 1 (3%) 1 (3%) 

28 (93%) 

1 (3%) 

20 (67%) 13 (43%) 

27 (90%) 18 (60%) 27 (90%) 

5 (17%) 

24 (80%) 15 (50%) 

17 (57%) 

1 (3%) 

3 (10%) 2 (7%) 2 (7%) 

23 (77%) 

3 (10%) 

1 (3%) 

1 (3%) 

a Number of animals examined microscopically at site and number of animals with lesion 
120 Skin Paint Studies, NTP TR 441 
121 

SKIN PAINT STUDIES 

IN THE COMPARATIW INITIATION/PROMOTION 

SUMMARY OF SKIN LESIONS 

APPENDIX B 

IN MALE AND FEMALE SWISS (CD-l@) MICE 

TABLE B3a 

TABLE B2d 

TABLE B2c 

TABLE B2b 

TABLE B2a 

TABLE Bld 

TABLE Blc 

TABLE Blb 

TABLE Bla 

TABLE B3b 

TABLE B3c 

TABLE B3d 

Skin Paint Studies: Study Design B 

Female Swiss (CD-1") Mice in the Comparative InitiatiodPromotion 

Summary of the Incidence of Nonneoplastic Skin Lesions in 

Skin Paint Studies: Study Design A 

Female Swiss (CD-la) Mice in the Comparative Initiation/Promotion 

Summary of the Incidence of Nonneoplastic Skin Lesions in 

Skin Paint Studies: Study Design B 

Male Swiss (CD-l@) Mice in the Comparative InitiatiodPromotion 

Summary of the Incidence of Nonneoplastic Skin Lesions in 

Skin Paint Studies: Study Design A 

Male Swiss (CD-la) Mice in the Comparative InitiationPromotion 

Summary of the Incidence of Nonneoplastic Skin Lesions in 

InitiationlPromotion Skin Paint Studies: Study Design B 

Overview of the Sensitivity of Female Swiss (CD-le) Mice in the Comparative 

InitiatiodPromotion Skin Paint Studies: Study Design A 

Overview of the Sensitivity of Female Swiss (CD-la) Mice in the Comparative 

InitiationlPromotion Skin Paint Studies: Study Design B 

Overview of the Sensitivity of Male Swiss (CD-la) Mice in the Comparative 

Initiatioflromotion Skin Paint Studies: Study Design A 

Overview of the Sensitivity of Male Swiss (CD-la) Mice in the Comparative 

in the Comparative InitiatiodPromotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Skin Neoplasms in Female Swiss (CD-1") Mice 

in the Comparative InitiatiodPromotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Skin Neoplasms in Female Swiss (CD-la) Mice 

in the Comparative InitiatiodPromotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Skin Neoplasms in Male Swiss (CD-la) Mice 

in the Comparative InitiationlPromotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Skin Neoplasms in Male Swiss (CD-la) Mice 

....... 

....... 

....... 

....... 

....................... 

....................... 

....................... 

....................... 

...................................... 

...................................... 

...................................... 

...................................... 

126 

124 

122 

130 

130 

128 

131 

131 

132 

134 

136 

138 
122 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Skin Neoplasms in Male Swiss (CD-lm) Mice in the Comparative InitiatiodPromotion 

TABLE Bla 

Acetone/ 0.25 pg DMBN 0.25 pg DMBN 2.5 pg DMBN 2.5 pg DMBN 2.5 pg DMBN 

Acetone Acetone 5 pg TPA Acetone 2.5 pg DMBA 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

2 

1 

30 30 

4 

6 

30 

3 

2 

Survivors 

Terminal sacrifice 27 25 20 

30 30 30 

1 

4 

7 

7 

8 

29 19 15 

Animals examined microscopically 30 30 30 30 30 30 

Site of application-mass, 

Squamous cell papilloma 

Squamous cell carcinoma 

Skin 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

basal cell adenoma 

Site of application-mass, 

Site of application-mass, sarcoma 

keratoacanthoma, multiple 

Subcutaneous tissue, 

lymphoma malignant lymphocytic 

Subcutaneous tissue, control, 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Subcutaneous tissue, site of 

Subcutaneous tissue, sarcoma 

hemangiosarcoma 

malignant lymphocytic 

application-mass, lymphoma 

(30) (30) (30) 

1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

2 (7%) 11 (37%) 

6 (20%) 6 (20%) 

4 (13%) 

12 (40%) 6 (20%) 

11 (37%) 

1 (3%) 

1 (3%) 

1 (3%) 

9 (30%) 

7 (23%) 

1 

Total animals with malignant neoplasms 1 

Total animals with benign neoplasms 

1 

Total animals with primary neoplasmsb 1 

Neoplasm Summary 

1 

1 

1 

1 

17 

16 

18 

16 

1 

1 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

23 

23 

10 

9 

33 

24 

12 

11 

18 

17 

30 

21 
Lesions in Male and Female Swiss (CD-la) Mice 123 

Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Skin Neoplasms in Male Swiss (CD-la) Mice in the Comparative Initiation/Promotion 

TABLE Bla 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

27 

1 

2 

30 30 

28 

1 

1 

30 

15 

2 

13 

30 

9 

SuMvors 

21 

Animals examined microscopically 30 30 30 30 

30 30 

6 

3 

2 

24 25 

30 30 

Site of application-mass, 

Squamous cell papilloma 

Squamous cell carcinoma 

Skin (continued) 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

basal cell adenoma 

Site of application-mass, 

Site of application-mass, sarcoma 

keratoacanthoma, multiple 

Subcutaneous tissue, 

lymphoma malignant lymphocytic 

Subcutaneous tissue, control, 

squamous cell papilloma, multiple 1 (3%) 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Subcutaneous tissue, site of 

Subcutaneous tissue, sarcoma 

hemangiosarcoma 

malignant lymphocytic 

application-mass, lymphoma 

(30) (30) (30) (30) (30) (30) 

1 (3%) 

2 (7%) 2 (7%) 

1 (3%) 

1 (3%) 

1 (3%) 1 (3%) 

1 (3%) 

10 (33%) 

4 (13%) 

4 (13%) 6 (20%) 

1 (3%) 13 (43%) 1 (3%) 

1 (3%) 

1 (3%) 

Neoplasm Summary (continued) 

3 

Total animals with benign eoplasms 3 

3 

Total animals with primary neoplasms 3 

Total animals with malignant neoplasms 

3 

3 

3 

3 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

1 

1 

1 

1 

15 

14 

18 

17 

33 

21 

2 

2 

8 

8 

10 

10 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primaly neoplasms: alt neoplasms except metastatic neoplasms 
124 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Skin Neoplasms in Male Swiss (CD-la) Mice in the Comparative Initiatioflromotion 

TABLE Blb 

Acetone/ 5 pg TPN 100 pg MNNG/ 100 pg MNNG/ 100 pg MNNG/ 100 fig MNNG/ 

Acetone 5 pg TPA Acetone 100 pg MNNG 20 mg BPO 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 3 

30 

17 

2 

11 

30 

28 

1 

1 

30 

survivors 

27 

Animals examined microscopically 30 30 30 

30 30 30 

1 

15 

1 

3 

3 

17 

14 26 10 

30 30 30 

Squamous cell carcinoma, 

Squamous cell carcinoma 

skin (30) (30) 

30) (30) (30) (30) 

1 (3%) 

metastatic, skin 1 (3%) 

Subcutaneous tissue, sarcoma 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-no mass, 

Control, lymphoma malignant 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, hemangiosarcoma 

Subcutaneous tissue, site of 

application-mass, hemangioma 

Subcutaneous tissue, site of 

application-no mass, fibroma 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 

squamous cell papilloma 

squamous cell carcinoma, multiple 

squamous cell carcinoma 

keratoacanthoma, multiple 

keratoacanthoma 

lymphoma malignant lymphocytic 

lymphocytic 

1 (3%) 

3 (10%) 

1 (3%) 

1 (3%) 

6 (20%) 

5 (17%) 

9 (30%) 

1 (3%) 

6 (20%) 

1 (3%) 

1 (3%) 

5 (17%) 

4 (13%) 

1 (3%) 

Neoplasm Summary 

Total animals with metastatic neoplasms 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasmsb 

Total metastatic neoplasms 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

1 

1 

21 

21 

7 

7 

28 

23 

2 

2 

9 

9 

11 

10 
Lesions in Male and Female Swiss (CD-lm) Mice 125 

Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Skin Neoplasms in Male Swiss (CD-l@) Mice in the Comparative InitiatiodPromotion 

TABLE Blb 

500 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 20 mg BPO/ 

Acetone 20 mg BPO Acetone 20 mg BPO 5 pg TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

30 

18 

3 

9 

30 

9 

2 

19 

30 

17 

1 

12 

Survivors 

Animals examined microscopically 30 30 30 

30 

5 

17 

8 

30 

30 30 

4 

22 

1 

9 

4 20 

30 30 

Squamous cell carcinoma 

Skin (continued) (30) (30) (30) (30) (30) (30) 

1 (3%) 1 (3%) 

Subcutaneous tissue, sarcoma 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-no mass, 

Control, lymphoma malignant 

Squamous cell carcinoma, 

Subcutaneous tissue, site of 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 

squamous cell papilloma 

squamous cell carcinoma, multiple 

squamous cell carcinoma 

keratoacanthoma, multiple 

keratoacanthoma 

lymphoma malignant lymphocytic 

lymphocytic 

metastatic, skin 

1 (3%) 

application-no mass, fibroma 

1 (3%) 2 (7%) 

1 (3%) 

3 (10%) 

1 (3%) 

4 (13%) 

3 (10%) 

1 (3%) 

4 (13%) 

1 (3%) 

2 (7%) 

6 (20%) 

3 (10%) 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, hemangiosarcoma 

Subcutaneous tissue, site of 

application-mass, hemangioma 1 (3%) 

11 (37%) 

1 (3%) 

2 (7%) 4 (13%) 7 (23%) 9 (30%) 

Neoplasm Summary (continued) 

Total animals with metastatic neoplasms 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primaxy neoplasms 

Total metastatic neoplasms 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 3 

3 

4 

4 

1 

1 

5 

5 

13 

12 

2 

2 

15 

13 

3 

3 

13 

13 

4 

4 

17 

16 

17 

17 

12 

10 

29 

20 

1 

1 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 
126 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Skin Neoplasms in Female Swiss (CD-la) Mice in the Comparative Initiatioflromotion 

TABLE Blc 

Acetone/ 0.25 pg DMBN 0.25 pg DMBN 2.5 pg DMBA/ 2.5 pg DMBN 2.5 pg DMBN 

Acetone Acetone 5 pg TPA Acetone 2.5 pg DMBA 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

Died last week of study 

30 30 30 

1 

1 

2 

6 

1 

SuMvors 

27 

1 

29 22 

Animals examined microscopically 30 30 30 

30 30 

3 

7 

1 

30 

29 

30 

20 , 

30 

3 

7 

19 

1 

30 

Sebaceous gland, site of 

Squamous cell papilloma 

Skin 

Site of application-no mass, 

(30) (30) (30) (30) (30) (30) 

application-mass, adenoma 1 (3%) 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, fibrosarcoma 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma, multiple 

3 (10%) 

5 (17%) 

2 (7%) 

5 (17%) 

10 (33%) 

7 (23%) 

16 (53%) 

1 (3%) 1. (3%) 

1 (3%) 

4 (13%) 

8 (27%) 

3 (10%) 3 (10%) 

5 (17%) 12 (40%) 

1 (3%) 

1 (3%) 

Neoplasm Summary 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasms b 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

18 

16 

25 

18 

7 

7 

24 

23 

9 

8 

33 

25 

13 

13 

18 

15 

31 

20 
Lesions in Male and Female Swiss (CD-la) Mice 127 

Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Skin Neoplasms in Female Swiss (CD-1") Mice in the Comparative Initiatioflromotion 

TABLE Blc 

2.5 pg DMBA/ 5 pg TPN 25 pg DMBA/ 25 pg DMBA/ 25 pg DMBA/ 20 mg BPO/ 

20 mg BPO 5 pg TPA Acetone 5 pg TPA 20 mg BPO 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

Died last week of study 

30 

1 

1 

SuMvors 

28 

Animals examined microscopically 30 

30 30 30 30 30 

1 

4 

1 

23 

1 

1 

28 

1 

4 

10 

16 30 30 

30 30 30 30 30 

Sebaceous gland, site of 

Squamous cell papilloma 

Skin (continued) 

squamous cell carcinoma 

Site of application-no mass, 

application-mass, adenoma 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

application-mass, sarcoma 

Subcutaneous tissue, site ol 

application-mass, fibrosarcoma 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 1 (3%) 

Site of application-mass, 

squamous cell papiloma 4 (13%) 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma, multiple 

(30) (30) 

1 (3%) 

1 (3%) 

5 (17%) 

1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

11 (37%) 

18 (60%) 

4 (13%) 

6 (20%) 

2 (7%) 

3 (10%) 

2 (7%) 

Neoplasm Summary (continued) 

5 

Total animals with benign eoplasms 5 

5 

Total animals with primary neoplasms 5 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

1 

1 

1 

1 

19 

18 

23 

22 

42 

28 

~~ ~ ~~ ~~ ~~ 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 
128 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Skin Neoplasms in Female Swiss (CD-le) Mice in the Comparative Initiation/Promotion 

TABLE Bld 

Acetone/ 5 pg TPN 100 pg MNNG/ 100 pg MNNG/ 100 pg MNNG/ 500 kg MNNG/ 

Acetone 5 pg TPA Acetone 100 gg MNNG 5 pg TPA Acetone 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

27 

1 

2 

30 

2 

23 

1 

4 

30 

1 

30 

Other 

SuMvors 

29 

30 

2 

9 

19 

30 

2 

6 

22 

30 

1 

2 

1 

26 

Animals examined microscopically 30 28 30 30 30 29 

Site of application-no mass, 

Squamous cell papilloma 

Skin 

squamous cell carcinoma, multiple 

Site of application-no mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

Subcutaneous tissue, lymphoma 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Subcutaneous tissue, site of 

Subcutaneous tissue, sarcoma 

malignant lymphocytic 

(30) (28) (30) 

application-no mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, fibroma 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, hemangioma 

4 (14%) 

1 (4%) 

(30) (30) (29) 

1 (3%) 

1 (3%) 

1 (3%) 

11 (37%) 5 (17%) 

5 (17%) 8 (27%) 

4 (13%) 5 (17%) 

4 (13%) 

2 (7%) 

1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

4 (13%) 2 (7%) 

Neoplasm Summary 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasmsb 

Total malignant neoplasms 

Total benign neoplasms 

Total primaxy neoplasms 

19 

17 

10 

9 

29 

19 

15 

13 

21 

15 

6 

6 
Lesions in Male and Female Swiss (CD-la) Mice 129 

Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Skin Neoplasms in Female Swiss (CD-l@) Mice in the Comparative Initiatioflromotion 

TABLE Bld 

100 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 20 mg BPO/ 

20 mg BPO 20 mg BPO Acetone 20 mg BPO 5 pg TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

30 

20 

1 

9 

30 

15 

2 

13 

30 

17 

3 

10 

30 

1 

14 

2 

13 

30 30 

2 1 

Other 

sunivors 

28 29 

Animals examined microscopically 30 30 30 30 29 30 

Site of application-no mass, 

Squamous cell papilloma 

Skin (continued) 

squamous cell carcinoma, multiple 

Site of application-no mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

Subcutaneous tissue, lymphoma 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous &11 carcinoma 

Subcutaneous tissue, site of 

Subcutaneous tissue, sarcoma 

malignant lymphocytic 

application-no mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, fibroma 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, hemangioma 

(30) 

1 (3%) 

2 (7%) 

1 (3%) 

1 (3%) 

1 (3%) 

2 (7%) 2 (7%) 7 (24%) 

1 (3%) 1 (3%) 1 (3%) 

3 (10%) 3 (10%) 3 (10%) 3 (10%) 

2 (7%) 

6 (20%) 

1 (3%) 2 (7%) 1 (3%) 

1 (3%) 

3 (10%) 5 (17%) 6 (20%) 3 (10%) 

Neoplasm Summary (continued) 

3 

Total animals with benign eoplasms 3 

3 

Total animals with primary neoplasms 3 

Total animals with malignant neoplasms 

7 

6 

7 

7 

14 

13 

10 

9 

2 

2 

12 

10 

13 

12 

2 

2 

15 

13 

9 

8 

9 

9 

18 

15 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 
130 Skin Paint Studies, NTP TR 441 

Study Design A 

Overview of the Sensitivity of Male Swiss (CD-lm) Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE B2a 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors Total Micea Mice with Tumorsb (Weeks) 

2.5 pg DMBU.5 pg DMBA 

25 pg DMBUO mg BPO 

2.5 pg DMBUO mg BPO 

25 pg DMBN5 pg TPA 

2.5 pg DMBA/5 pg TPA 

0.25 pg DMBN5 pg TPA 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

20 mg BPODO mg BPO 

5 pg TPN5 pg TPA 

Acetone/Acetone 

22/30 

6/30 

1/30 

28/30 

27/30 

18/30 

on0 

on0 

1/30 

on0 

5/30 

on0 

50.0 

34.6 

- 

42.9 

47.0 

52.0. 

17.6 

15.4 

27.7 

- 

1.30 

0.20 

0.03 

5.60 

4.63 

1.07 

0.00 

0.00 

0.03 

0.00 

0.17 

0.00 

1.77 

1.00 

1.00 

6.00 

5.15 

1.78 

0.00 

0.00 

1 .oo 

0.00 

1 .oo 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 

Study Design B 

Overview of the Sensitivity of Male Swiss (CD-b) Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE B2b 

With Tumors 

Number of Mice to Tumor 

Mean Time Average Number Average Number 

of Tumors/ of Tumors/ 

Total Micea Mice with Tumorsb (Weeks) 

100 pg MNNG/100 pg MNNG 

1,000 pg MNNGnO mg BPO 

500 pg MNNGDO mg BPO 

100 pg MNNGDO mg BPO 

1,000 pg MNNG/5 pg "PA 

100 pg MNNG/5 pg TPA 

1,000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

20 mg BP0/20 mg BPO 

5 pg TPN5 kg TPA 

Acetone/Acetone 

17/30 

13/30 

11/30 

1/30 

23/30 

16/30 

15/30 

7/30 

1/30 

on0 

4/30 

0/30 

35.8 

40.2 

10.9 

18.3 

38.0 

12.9 

25.3 

15.9 

14.9 

15.0 

- 

- 

0.97 

0.83 

0.63 

0.03 

1.87 

1.40 

0.73 

0.30 

0.03 

0.00 

0.20 

0.00 

1.71 

1.92 

1.73 

1.00 

2.43 

2.63 

1.47 

1.29 

1.00 

0.00 

1.50 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 
Lesions in Male and Female Swiss (CD-la) Mice 131 

Study Design A 

Overview of the Sensitivity of Female Swiss (CD-le) Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE B2c 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors Total Micea Mice with Tumorsb (Week) 

2.5 pg DMBM.5 pg DMBA 

25 pg DMBUO mg BPO 

2.5 pg DMBAnO mg BPO 

25 pg DMBN5 pg TPA 

2.5 pg DMBN5 pg TPA 

0.25 pg DMBN5 cg TPA 

25 pg DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

20 mg BPODO mg BPO 

5 Kg TPN5 fig TPA 

AcetonelAcetone 

40.1 

39.2 

51.0 

16.0 

18.0 

26.5 

- 

1.27 

0.30 

0.03 

7.10 

3.77 

1.43 

0.00 

0.00 

0.00 

0.00 

0.13 

0.00 

1.90 

1.80 

1.00 

7.34 

4.35 

2.39 

0.00 

0.00 

0.00 

0.00 

1.00 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 

Study Design B 

Overview of the Sensitivity of Female Swiss (CD-la) Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE B2d 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors (Weeks) Total Micea Mice with Tumorsb 

100 pg MNNG/100 pg MNNG 

1,000 pg MNNGm mg BPO 

500 pg MNNGDO mg BPO 

100 pg MNNGDO mg BPO 

1,000 pg MNNG/5 pg TPA 

100 pg MNNG/5 pg TPA 

1,OOO pg MNNG/Acetone 

500 pg MNNWAcetone 

100 pg MNNWAcetone 

20 mg BPODO mg BPO 

5 pg TPN5 cg TPA 

AcetondAcetone 

19/30 

19/30 

13/30 

3/30 

21/30 

21/30 

9/30 

6/30 

on0 

0/30 

8/30 

0/30 

34.1 

- 

38.7 

17.8 

8.4 

31.0 

18.8 

21.1 

11.7 

18.0 

- 

0.87 

0.90 

0.62 

0.13 

1.23 

2.27 

0.40 

0.27 

0.00 

0.00 

0.33 

0.00 

1.37 

1.42 

1.42 

1.33 

1.76 

3.24 

1.33 

1.33 

0.00 

0.00 

1.25 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 
132 Skin Paint Studies, NTP TR 441 

Initiatioflromotion Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male Swiss (CD-lm) Mice in the Comparative 

TABLE B3a 

Acetone/ 0.25 pg DMBN 0.25 pg DMBN 2.5 pg DMBN 2.5 pg DMBN 2.5 pg DMBN 

Acetone Acetone 5 pg TPA Acetone 2.5 pg DMBA 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

27 

2 

1 

30 

25 

3 

2 

30 

20 

4 

6 

30 

1 

30 

survivors 

29 

Animals examined microscopically 30 30 30 30 

30 

4 

7 

19 

30 

30 

7 

8 

30 

15 

acanthosis 

Site of application-mass, 

ulcer, multiple 

Site of application-no mass, 

ulcer 5 (17%) 9 

%) 

Site of application-no mass, 

inflammation, chronic active 4 (13%) 7 (23%) 30 (100%) 5 (17%) 25 (83%) 29 (97%) 

Site of application-no mass, 

alopecia 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-no mass, cyst 

Hair follicle, site of 

Skin (30) (30) (30) (301 (30) (30) 

4 (13%) 8 (27%) 30 (100%) 4 (13%) 26 (87%) 30 (100%) 

1 (3%) 2 (7%) 10 (33%) 

1 (3%) 11 (37%) 

1 (3%) 

1 (3%) 8 (27%) 
Lesions in Male and Female Swiss (CD-l@) Mice 133 

InitiatiodPromotion Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male Swiss (CD-lm) Mice in the Comparative 

TABLE B3a 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

27 

1 

2 

30 

9 

30 

28 

1 

1 

30 

SuMvors 

21 

Animals examined microscopically 30 30 30 

30 30 

2 

13 6 

15 24 

30 30 

30 

3 

2 

30 

25 

acanthosis 

Site of application-mass, 

ulcer, multiple 

Site of application-no mass, 

ulcer 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

alopecia 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-no mass, cyst 

Hair follicle, site of 

Skin (continued) (30) (30) (30) (30) (30) (30) 

1 (3%) 1 (3%) 

25 (83%) 30 (100%) 5 (17%) 30 (100%) 29 (97%) 27 (90%) 

1 (3%) 

7 (23%) 8 (27%) 5 (17%) 4 (13%) 

5 (17%) 7 (23%) 7 (23%) 6 (20%) 

24 (80%) 30 (100%) 5 (17%) 30 (100%) 

2 (7%) 

28 (93%) 28 (93%) 

2 (7%) 

1 (3%) 

a Number of animals examined microscopically at site and number of animals with lesion 
134 Skin Paint Studies, NTP TR 441 

Initiation/Promotion Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male Swiss (CD-1") Mice in the Comparative 

TABLE B3b 

Acetone/ 5 pg TPN 100 pg MNNG/ 100 pg MNNG/ 100 pg MNNG/ 100 pg MNNG/ 

Acetone 5 pg TPA Acetone 100 pg MNNG 20 mg BPO 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 3 

30 

2 

11 

30 

1 

1 

30 

14 

1 

15 

30 

Survivors 

27 17 28 

30 

1 

3 

26 

30 

3 

17 

10 

Animals examined microscopically 30 30 30 30 30 30 

acanthosis 

Site of application-mass, 

ulcer 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

hemorrhage 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-mass, hyperplasia 

Sebaceous gland, site of 

application-no mass, cyst 

Hair follicle, site of 

Skin 

Site of application-mass, 

hyperplasia, mast cell 

Site of application-mass, 

ulcer 

(30) (30) (30) (30) (30) (30) 

4 (13%) 30 (100%) 7 (23%) 29 (97%) 22 (73%) 29 (97%) 

2 (7%) 

2 (7%) 

8 (27%) 

24 (80%) 

1 (3%) 1 (3%) 2 (7%)

3 (10%) 

2 (7%) 

9 (30%) 7 (23%) 

19 (63%) 

11 (37%) 

11 (37%) 28 (93%) 
Lesions in Male and Female Swiss (CD-l@) Mice 135 

InitiatiodPromotion Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male Swiss (CD-l@) Mice in the Comparative 

TABLE B3b 

500 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 20 mg BPO/ 

Acetone 20 mg BPO Acetone 20 mg BPO 5 pg TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 

1 

12 

30 

3 

9 

30 30 30 

2 

19 

5 

17 

SuMvors 

Terminal sacrifice 17 18 9 8 

Animals examined microscopically 30 30 30 30 

30 30 

4 

22 

1 

9 

30 

4 

30 

20 

acanthosis 

Site of application-mass, 

ulcer 9 (30%) 

Site of application-no mass, 

inflammation, chronic active 13 (43%) 

Site of application-no mass, 

hemorrhage 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-mass, hyperplasia 

Sebaceous gland, site of 

application-no mass, cyst 

Hair follicle, site of 

Skin (continued) 

Site of application-mass, 

hyperplasia, mast cell 

Site of application-mass, 

ulcer 

(30) 

13 (43%) 

1 (3%) 

(30) (30) (30) 

1 (3%) 2 (7%) 

1 (3%) 

25 (83%) 16 (53%) 25 (83%) 28 (93%) 25 (83%) 

1 (3%) 

8 (27%) 9 (30%) 8 (27%) 

20 (67%) 21 (70%) 20 (67%) 25 (83%) 

1 (3%) 

3 (10%) 

15 (50%) 

10 (33%) 

2 (7%) 

1 (3%) 

a Number of animals examined microscopically at site and number of animals with lesion 
136 Skin Paint Studies, NTP TR 441 

Initiatioflromotion Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female Swiss (CD-lm) Mice in the Comparative 

TABLE B3c 

Acetone/ 0.25 pg DMBN 0.25 pg DMBN 2.5 pg DMBN 2.5 pg DMBA/ 2.5 pg DMBA/ 

Acetone Acetone 5 pg TPA Acetone 2.5 pg DMBA 5 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

Died last week of study 

30 30 30 

27 

1 

1 

1 

2 

6 

1 

SuMvors 

29 22 

30 30 

3 

7 

1 

29 20 

30 

3 

7 

19 

1 

Animals examined microscopically 30 30 30 30 30 30 

acanthosis 

Site of application-mass, 

ulcer, multiple 

Site of application-no mass, 

ulcer 

Site of application-no mass, 

inflammation, chronic active 9 (30%) 5 (17%) 29 (97%) 

Site of application-no mass, 

acanthosis 5 (17%) 29 

Site of application-no mass, 

application-no mass, cyst 

Hair follicle, site of 

Skin (30) (30) (30) (30) (30) (30) 

1 (3%) 

10 (33%) 

3 (10%) 

5 (17%) 

27 (90%)30 (100%) 

25 (83%) 30 (100%) 

1 (3%) 

1 (3%) 3 (10%) 

5 (17%) 

1 (3%) 

2 (7%) 5 (17%) 
Lesions in Male and Female Swiss (CD-la) Mice 137 

Initiatioflromotion Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female Swiss (CD-la) Mice in the Comparative 

TABLE B3c 

2.5 pg DMBN 5 pg TPN 25 Kg DMBN 25 pg DMBN 25 pg DMBN 20 mg BPO/ 

20 mg BPO 5 pg TPA Acetone 5 pg TPA 20 mg BPO 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 

Accidental death 

30 30 

1 

4 

1 

30 30 30 

1 

1 

4 

10 

1 

30 

Sulvivors 

Terminal sacrifice 

Died last week of study 

23 

1 

28 

1 

28 16 30 30 

Animals examined microscopically 30 30 30 30 30 30 

acanthosis 

Site of application-mass, 

ulcer,  5 (17%) 

Site of application-no mass, 

ulcer 4 (13%) 4 (13%) 

Site of application-no mass, 

inflammation, chronic active 30 (100%) 29 (97%) 5 (17%) 30 (100%) 

Site of application-no mass, 

acanthosis 30 (100%) 30 (100%) 30 (100%) 

Site of application-no mass, 

application-no mass, cyst 

Hair follicle, site of 

Skin (continued) (30) (30) 

30) 

1 (3%) 

28 (93%) 

28 (93%) 

30 (100%) 

30 (100%) 

2 (7%) 

1 (3%) 

1 (3%) 

11 (37%) 1 (3%) 

1 (3%) 

a Number of animals examined microscopically at site and number of animals with lesion 
138 Skin Paint Studies, NTP TR 441 

InitiatiodPromotion Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female Swiss (CD-la) Mice in the Comparative 

TABLE B3d 

Acetone/ 5 pg TPN 100 pg MNNG/ 100 pg MNNG/ 100 pg MNNG/ 500 pg MNNG/ 

Acetone 5 pg TPA Acetone 100 pg MNNG 5 pg TPA Acetone 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

27 

1 

2 

30 

2 

23 

1 

4 

30 30 

19 

2 

9 

30 

1 

Other 

SuMvors 

29 

Animals examined microscopically 30 28 30 30 

30 30 

2 

6 

1 

2 

22 

1 

26 

30 29 

cellular, mixed cell 

Subcutaneous tissue, infiltration 

ulcer 

Site of application-mass, 

acanthosis 

Site of application-mass, 

ulcer 

Site of application-no mass, 

inflammation, chronic active 

Site of application-no mass, 

hemorrhage 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-no mass, cyst 

Hair follicle, site of 

Skin (30) (28) (30) (30) (30) (29) 

2 (7%) 

2 (7%) 27 (96%) 7 (23%) 27 (90%) 25 (83%) 14 (48%) 

1 (3%) 

1 (3%) 

1 (3%) 

7 (25%) 7 (23%) 

25 (89%) 6 (20%) 27 (90%) 28 (93%) 13 (45%) 

1 (3%) 8 (27%) 

1 (3%) 1 (3%) 1 (3%) 

2 (7%) 

1 (4%) 
Lesions in Male and Female Swiss (CD-l@) Mice 139 

InitiatiodPromotion Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female Swiss (CD-lm) Mice in the Comparative 

TABLE B3d 

100 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 1,000 pg MNNG/ 20 mg BPO/ 

20 mg BPO 20 mg BPO Acetone 20 mg BPO 5 fig TPA 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 2 

30 

20 

1 

9 

30 

15 

2 

13 

30 

Other 

survivors 

28 

30 30 30 

3 

10 

17 

2 

13 

1 

14 

1 

29 

Animals examined microscopically 30 30 30 30 29 30 

ulcer 

Site of application-mass, 

acanthosis 

Site of application-mass, 

ulcer 

Site of application-no mass, 

inflammation, chronic active 26 (87%) 

Site of application-no mass, 

hemorrhage 

Site of application-no mass, 

acanthosis 

Site of application-no mass, 

application-no mass, cyst 

Hair follicle, site of 

Skin (continued) (30) (30) (30) (30) (29) (30) 

2 (7%) 1 (3%) 

28 (93%) 28 (93%) 22 (73%) 24 (80%) 26 (90’70) 21 (70%) 

26 (87%) 

6 (20%) 5 (17%) 9 (31%) 

19 (63%) 20 (67%) 27 (93%) 16 (53%) 

2 (7%) 2 (7%) 

1 (3%) 

Subcutaneous tissue, infiltration 

cellular, mixed cell 1 (3%) 

a Number of animals examined microscopically at site and number of animals with lesion 
140 Skin Paint Studies, NTP TR 441 
141 

IN THE COMPARATIVE INITIATION/PROMOTION 

IN MALE AND FEMALE SENCAR MICE 

SUMMARY OF SKIN LESIONS 

APPENDIX C 

SKIN PAINT STUDIES 

TABLE C3f 

TABLE C3e 

TABLE C3d 

TABLE C3c 

TABLE C3b 

TABLE C3a 

TABLE C2d 

TABLE C2c 

TABLE C2b 

TABLE C2a 

TABLE Clf 

TABLE Cle 

TABLE Cld 

TABLE Clc 

TABLE Clb 

TABLE Cla 

in the Comparative InitiationPromotion Skin Paint Studies: Study Restart 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female SENCAR Mice 

in the Comparative InitiationlPromotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female SENCAR Mice 

in the Comparative InitiatiodPromotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female SENCAR Mice 

in the Comparative Initiatioflromotion Skin Paint Studies: Study Restart 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male SENCAR Mice 

in the Comparative Initiatioflromotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male SENCAR Mice 

in the Comparative InitiatiorVPromotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male SENCAR Mice 

InitiatiorVPromotion Skin Paint Studies: Study Design B 

Overview of the Sensitivity of Female SENCAR Mice in the Comparative 

InitiatiordPromotion Skin Paint Studies: Study Design A 

Overview of the Sensitivity of Female SENCAR Mice in the Comparative 

Initiatioflromotion Skin Paint Studies: Study Design B 

Overview of the Sensitivity of Male SENCAR Mice in the Comparative 

Initiatioflromotion Skin Paint Studies: Study Design A 

Overview of the Sensitivity of Male SENCAR Mice in the Comparative 

in the Comparative Initiatioflromotion Skin Paint Studies: Study Restart 

Summary of the Incidence of Skin Neoplasms in Female SENCAR Mice 

in the Comparative InitiatiorVPromotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Skin Neoplasms in Female SENCAR Mice 

in the Comparative InitiatiorVPromotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Skin Neoplasms in Female SENCAR Mice 

in the Comparative Initiatioflromotion Skin Paint Studies: Study Restart 

Summary of the Incidence of Skin Neoplasms in Male SENCAR Mice 

in the Comparative Initiatioflromotion Skin Paint Studies: Study Design B 

Summary of the Incidence of Skin Neoplasms in Male SENCAR Mice 

in the Comparative Initiatioflromotion Skin Paint Studies: Study Design A 

Summary of the Incidence of Skin Neoplasms in Male SENCAR Mice 

166 

164 

162 

160 

158 

156 

155 

155 

154 

154 

152 

150 

148 

146 

144 

....... 142 

....... 

........ 

....... 

....... 

........ 

....................... 

....................... 

....................... 

....................... 

....... 

....... 

........ 

....... 

....... 

........ 
142 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Skin Neoplasms in Male SENCAR Mice in the Comparative Initiatioflromotion 

TABLE Cla 

Acetone 

Acetonel 0.25 pg DMBN 2.5 pg DMBN 2.5 pg DMBN 

Acetone I Acetone 2.5 pg DMBA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

SuMvors 

30 

1 

1 

28 

30 

2 

1 

27 

29 

1 

1 

27 

30 

4 

15 

11 

Animals examined microscopically 30 30 29 30 

Site of application-mass, 

Squamous cell papilloma 

skin 

Subcutaneous tissue, site of 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

application-mass, fibrosarcoma 

squamous cell papilloma, multiple 

squamous cell papilloma 

squamous cell carcinoma, multiple 

squamous cell carcinoma 

keratoacanthoma, multiple 

keratoacanthoma 3 (10%) 

1 (3%) 

5 (17%) 

13 (43%) 

14 (47%) 

Neoplasm Summary 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasmsb 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

27 

27 

11 

9 

38 

28 
Lesions in Male and Female SENCAR Mice 143 

Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Skin Neoplasms in Male SENCAR Mice in the Comparative Initiatioflromotion 

TABLE Cla 

2.5 pg DMBN 25 pg DMBN 25 pg DMBN 20 mg BPO/ 

20 mg BPO Acetone 20 mg BPO 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

23 

1 

6 

30 

SuMvors 

31 

30 

1 

30 

1 

12 

17 

30 

2 

5 

23 

Animals examined microscopically 30 31 30 30 

Site of application-mass, 

Squamous cell papilloma 

Skin (continued) 

Subcutaneous tissue, site of 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

Site of application-mass, 

(30) (31) 

application-mass, fibrosarcoma 

squamous cell papilloma, multiple 

squamous cell papilloma 

squamous cell carcinoma, multiple 

squamous cell carcinoma 

keratoacanthoma, multiple 

keratoacanthoma 

8 (27%) 

1 (3%) 

1 (3%) 

10 (33%) 

2 (7%) 

3 (10%) 

(30) (30) 

3 (10%) 

1 (3%) 

1 (3%) 

5 (17%) 

6 (20%) 

5 (17%) 

8 (27%) 

Neoplasm Summary (continued) 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasms 

11 

11 

14 

13 

25 

19 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

14 

14 

15 

12 

29 

18 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 
144 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Skin Neoplasms in Male SENCAR Mice in the Comparative Initiation/Promotion 

TABLE Clb 

Acetonel 100 pg MNNG/ 500 pg MNNGI 1,000 pg MNNGI 100 pg MNNG/ 

Acetone Acetone Acetone Acetone 100 pg MNNG 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Died last week of study 

Natural deaths 

Moribund 

30 

1 

Survivors 

29 

2 

30 

28 

4 

10 

30 

16 

3 

20 

30 

7 

3 

23 

30 

4 

Animals exmined microscopically 30 30 30 30 30 

Sebaceous gland, site of 

Squamous cell carcinoma, multiple 

Squamous cell carcinoma 

Skin 

squamous cell carcinoma 

Site of application-no mass, 

application-mass, adenoma 

Site of application-mass, 

basal cell adenoma 

Site of application-mass, 

Site of application-mass, 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma 

Subcutaneous tissue, site of 

Subcutaneous tissue, sarcoma 

trichoepithelioma 

application-no mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, fibroma 

Subcutaneous tissue, site of 

application-mass, sarcoma, multiple 

Subcutaneous tissue, site of application-mass, 

squamous cell carcinoma 

1 (3%) 

(30) 

2 (7%) 

1 (3%) 

5 (17%) 

1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

4 (13%) 

1 (3%) 

1 (3%) 

6 (20%) 

5 (17%) 

8 (27%) 

19 (63%) 

2 (7%) 

6 (20%) 

2 (7%) 2 (7%) 

1 (3%) 

2 (7%) 

3 (10%) 1 (3%) 

1 (3%) 

Neoplasm Summary 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primaly neoplasmsb 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

13 

13 

4 

4 

17 

14 

14 

14 

8 

8 

22 

18 

30 

27 

8 

8 

38 

28 
Lesions in Male and Female SENCAR Mice 145 

Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Skin Neoplasms in Male SENCAR Mice in the Comparative Initiatioflromotion 

TABLE Clb 

20 mg BPO/ 100 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 

20 mg BPO 20 mg BPO 20 mg BPO 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 3 

30 

SuMvors 

Terminal sacrifice 

Died last week of study 

26 

1 

Animals examined microscopically 30 

30 

2 

30 

28 

30 

3 

11 

30 

16 

30 

3 

21 

6 

30 

1 (3%) 6 (20%) 

1 (3%) 

1 (3%) 

2 (7%) 

2 (7%) 

1 (3%) 

Sebaceous gland, site of 

Squamous cell carcinoma, multiple 

Squamous cell carcinoma 

Skin (continued) 

squamous cell carcinoma 

Site of application-no mass, 

application-mass, adenoma 

Site of application-mass, 

basal cell adenoma 

Site of application-mass, 

Site of application-mass, 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma 

Subcutaneous tissue, site of 

Subcutaneous tissue, sarcoma 

trichoepithelioma 

application-no mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, fibroma 

Subcutaneous tissue, site of 

squamous cell carcinoma 

Subcutaneous tissue, site of application-mass, 

application-mass, sarcoma, multiple 

(30) (30) (30) (30) 

1 (3%) 

3 (10%) 

9 (30%) 

4 (13%) 

3 (10%) 

1 (3%) 

15 (50%) 

3 (10%) 

6 (20%) 

1 (3%) 

5 (17%) 

1 (3%) 

5 (17%) 

Neoplasm Summary (continued) 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasms 

1 

1 

1 

1 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

2 

2 

10 

9 

12 

11 

15 

14 

12 

11 

27 

18 

24 

23 

8 

8 

32 

27 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 
146 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Restarta 

Summary of the Incidence of Skin Neoplasms in Male SENCAR Mice in the Comparative Initiatioflromotion 

TABLE Clc 

Acetone1 0.25 pg DMBN 2.5 pg DMBA/ 25 pg DMBA/ 

Acetone 1 pg TPA 1 pg TPA 1 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

SuMvors 

1 

30 

29 

30 30 

2 

22 

6 

22 

6 2 

30 

4 

25 

1 

Animals examined microscopically 30 30 30 30 

Skin 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

keratoacanthoma, multiple 

Site of application-mass, 

basal cell carcinoma 

Site of application-mass, 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-no mass, sarcoma 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 4 (13%) 

1 (3%) 

3 (10%) 

2 (7%) 

7 (23%) 

2 (7%) 

5 (17%) 

4 (13%) 

2 (7%) 

16 (53%) 

3 (10%) 

5 (17%) 

4 (13%) 

Neoplasm Summary 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasmsb 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

6 

6 

5 

5 

11 

8 

9 

9 

9 

9 

18 

14 

19 

19 

11 

9 

30 

21 
Lesions in Male and Female SENCAR Mice 147 

Skin Paint Studies: Study Restart (continued) 

Summary of the Incidence of Skin Neoplasms in Male SENCAR Mice in the Comparative Initiatioflromotion 

TABLE Clc 

1 kg TPN 100 pg MNNG/ 1,000 pg MNNG/ 

1 pg TPA 1 pg TPA 1 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

SuMvors 

Animals examined microscopically 

30 

4 

17 

9 

30 

30 

2 

20 

8 

30 

30 

3 

26 

30 

1 

Skin (continued) 

Site of application-mass, 

keratoacanthoma 

Site of application-mass, 

keratoacanthoma, multiple 

Site of application-mass, 

basal cell carcinoma 

Site of application-mass, 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-no mass, sarcoma 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 1 (3%) 

3 (10%) 

1 (3%) 

2 (7%) 

3 (10%) 

1 (3%) 

1 (3%) 

1 (3%) 

2 (7%) 

2 (7%) 

4 (13%) 

14 (47%) 

Neoplasm Summary (continued) 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasms 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 1 

1 

1 

1 

21 

20 

4 

4 

25 

20 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Prirnaly neoplasms: all neoplasms except metastatic neoplasms 
148 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Skin Neoplasms in Female SENCAR Mice in the Comparative Initiatioflromotion 

TABLE Cld 

Acetonel 0.25 pg DMBN 2.5 pg DMBN 2.5 pg DMBN 

Acetone Acetone Acetone 2.5 pg DMBA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

SuMvors 

Animals examined microscopically 

30 

1 

2 

27 

30 

1 

30 

30 

29 

1 

31 

31 

30 

30 

1 

19 

10 

30 

Sebaceous gland, site of 

Squamous cell papilloma 

Squamous cell carcinoma 

Skin 

application-mass, adenoma 

Site of application-mass, 

basal cell carcinoma 

Site of application-mass, 

application-mass, fibrosarcoma 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma, multiple 

Site of application-mass, 

keratoacanthoma 

3 (10%) 

12 (40%) 

17 (57%) 

1 (3%) 

1 (3%) 

1 (3%) 

6 (20%) 

1 (3%) 

Neoplasm Summary 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasmsb 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

31 

29 

11 

9 

42 

29 
Lesions in Male and Female SENCAR Mice 149 

Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Skin Neoplasms in Female SENCAR Mice in the Comparative Initiation/Promotion 

TABLE Cld 

2.5 Kg DMBN 25 fig DMBN 25 fig DMBN 20 mg BPOI 

20 mg BPO Acetone 20 mg BPO 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

21 

1 

8 

30 

Survivors 

Animals examined microscopically 30 

1 

29 

29 

28 

30 

1 

6 

23 

30 

2 

30 

30 

28 

~~ ~ ~ ~~ ~~~ 

Sebaceous gland, site of 

Squamous cell papilloma 

Squamous cell carcinoma 

Skin (continued) 

application-mass, adenoma 

Site of application-mass, 

basal cell carcinoma 

Site of application-mass, 

application-mass, fibrosarcoma 

Subcutaneous tissue, site of 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of applicafion-mass, 

keratoacanthoma, multiple 

Site of application-mass, 

keratoacanthoma 4 (13%) 

5 (17%) 

6 (20%) 

3 (10%) 

11 (37%) 

1 (3%) 

1 (3%) 

1 (3%) 

4 (13%) 

1 (3%) 

9 (30%) 

2 (7%) 

8 (27%) 

8 (27%) 

Neoplasm Summary (continued) 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasms 

14 

14 

16 

14 

30 

22 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

12 

11 

22 

18 

34 

20 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: a11 neoplasms except metastatic neoplasms 
150 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Skin Neoplasms in Female SENCAR Mice in the Comparative Initiation/Promotion 

TABLE Cle 

Acetone/ 100 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 100 pg MNNG/ 

Acetone Acetone Acetone Acetone 100 pg MNNG 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

SuMvors 

30 

2 

1 

1 

26 

Animals exmined microscopically 30 

30 

30 

30 

30 

3 

5 

22 

30 

30 

6 

13 

11 

30 

30 

2 

25 

3 

30 

Site of application-no mass, 

Squamous cell carcinoma 

Skin 

squamous cell carcinoma 

Site of application-mass, 

basal cell adenoma 

Site of application-mass, 

malignant undifferentiated cell type 

Subcutaneous tissue, control, lymphoma 

undifferentiated cell type 

Subcutaneous tissue, lymphoma malignant 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

sarcoma 

Site of application-mass, 

keratoacanthoma 

undifferentiated cell type 

application-no mass, lymphoma malignant 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

(30) 

1 (3%) 

5 (17%) 

1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

1 (3%) 

14 (47%) 17 (57%) 

1 (3%) 2 (7%) 

3 (10%) 

1 (3%) 11 (37%) 

1 (3%) 

1 (3%) 

5 (17%) 

3 (10%) 

1 (3%) 

1 (3%) 4 (13%) 

Neoplasm Summary 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasmsb 

Total malignant neoplasms 

Total benign neoplasms 

Total primaly neoplasms 

20 

19 

4 

4 

24 

20 

32 

28 

9 

8 

41 

28 
Lesions in Male and Female SENCAR Mice 151 

Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Skin Neoplasms in Female SENCAR Mice in the Comparative Initiation/Promotion 

TABLE Cle 

20 mg BPOI 100 pg MNNGI 500 pg MNNGI 1,000 pg MNNGI 

20 mg BPO 20 mg BPO 20 mg BPO 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

30 

27 

2 

1 

survivors 

Animals examined microscopically 30 

30 

1 

30 

29 

30 

7 

30 

23 

30 

4 

12 

14 

30 

1 (3%) 

2 (7%) 

Site of application-no mass, 

Squamous cell carcinoma 

Skin (continued) 

squamous cell carcinoma 

Site of application-mass, 

basal cell adenoma 

Site of application-mass, 

malignant undilferentiated cell type 

Subcutaneous tissue, control, lymphoma 

undifferentiated cell type 

Subcutaneous tissue, lymphoma malignant 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

sarcoma 

Site of application-mass, 

keratoacanthoma 

malignant undifferentiated cell type 

application-no mass, lymphoma 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

(30) (3) 

3 (10%) 

3 (10%) 

1 (3%) 

1 (3%) 

1 (3%) 

5 (17%) 

4 (13%) 

1 (3%) 

d3) 

1 (3%) 

8 (27%) 

6 (20%) 

1 (3%) 

3 (10%) 4 (13%) 

Neoplasm Summary (continued) 

Total animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasms 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

2 

2 

8 

8 

10 

10 

9 

9 

5 

5 

14 

13 

13 

13 

7 

7 

20 

18 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: at1 neoplasms except metastatic neoplasms 
152 Skin Paint Studies, NTP TR 441 

Skin Paint Studies: Study Restarta 

Summary of the Incidence of Skin Neoplasms in Female SENCAR Mice in the Comparative Initiatioflromotion 

TABLE Clf 

Acetonel 0.25 pg DMBN 2.5 pg DMBN 25 pg DMBN 

Acetone 1 pg TPA 1 pg TPA 1 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

survivors 

Animals examined microscopically 

30 

2 

1 

30 

27 

30 

4 

18 

30 

8 

30 

5 

19 

30 

6 

30 

6 

23 

1 

30 

Site of application-mass, 

Squamous ceU papilloma 

Basal cell carcinoma 

skin 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous cell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma, multiple 

Site of application-mass, 

keratoacanthoma 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, sarcoma, multiple 

(29) 

2 (7%) 

6 (20%) 

3 (10%) 

10 (33%) 

1 (3%) 

1 (3%) 1 (3%) 

5 (17%) 

7 (23%) 

12 (40%) 

4 (13%) 

7 (23%) 

4 (13%) 

9 (30%) 

Neoplasm Summary 

Total animals with uncertain neoplasmsTotal animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasmsb 

benign or malignant 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

Total uncertain neoplasms 

10 

10 

12 

10 

2 

2 

15 

14 

13 

12 

28 

20 

19 

19 

15 

14 

34 

24 
Lesions in Male and Female SENCAR Mice 153 

Skin Paint Studies: Study Restart (continued) 

Summary of the Incidence of Skin Neoplasms in Female SENCAR Mice in the Comparative Initiation/Promotion 

TABLE Clf 

1 kg TPN 100 pg MNNGI 

1 pg TPA 1 Kg TPA 1 pg TPA 

1,000 pg MNNG/ 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

Survivors 

Animals examined microscopically 

30 

1 

19 

10 

30 

30 

2 

15 

13 

30 

30 

4 

24 

2 

30 

~ ~~ ~ ~ ~~~~~~ 

Site of application-mass, 

Squamous cell papilloma 

Basal cell carcinoma 

Skin (continued) 

squamous cell papilloma, multiple 

Site of application-mass, 

squamous cell papilloma 

Site of application-mass, 

squamous :ell carcinoma, multiple 

Site of application-mass, 

squamous cell carcinoma 

Site of application-mass, 

keratoacanthoma, multiple 

Site of application-mass, 

keratoacanthoma 

Subcutaneous tissue, site of 

application-mass, sarcoma 

Subcutaneous tissue, site of 

application-mass, sarcoma, multiple 

1 (3%) 

6 (20%) 

3 (10%) 

7 (23%) 

6 (20%) 

1 (3%) 

3 (10%) 

3 (10%) 

2 (7%) 

2 (7%) 

Neoplasm Summary (continued) 

Total animals with uncertain neoplasmsTotal animals with malignant neoplasms 

Total animals with benign neoplasms 

Total animals with primary neoplasms 

benign or malignant 

Total malignant neoplasms 

Total benign neoplasms 

Total primary neoplasms 

Total uncertain neoplasms 

10 

10 

16 

15 

6 

6 

12 

11 

5 

5 

17 

13 

a Number of animals examined microscopically at site and number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 
154 Skin Paint Studies, NTP TR 441 

Study Design A 

Overview of the Sensitivity of Male SENCAR Mice in the Comparative Initiatioflromotion Skin Paint Studies: 

TABLE C2a 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors (week) Total Micea Mice with Tumorsb 

2.5 pg DMBAn.5 pg DMBA 

25 pg DMBm mg BPO 

2.5 pg DMBm mg BPO 

25 pg DMBM pg TPA 

2.5 pg DMBM pg TPA 

0.25 pg DMBM pg TPA 

25 pg DMBNAcetone 

2.5 pg DMBA/Acetone 

0.25 pg DMBNAcetone 

20 mg BPOnO mg BPO 

1 pg TPN1 pg "PA 

AcetonelAcetone' 

Acetone/Acetone 

27/30 

22/30 

20/30 

24/30 

19/30 

12/30 

on1 

OD9 

0/30 

OB0 

2/30 

0/30 

0/30 - 

- 

16.0 

36.1 

31.0 

31.0 

14.5 

15.5 

17.2 

- 

2.53 

2.77 

2.40 

6.77 

3.03 

1.10 

0.00 

0.00 

0.00 

0.00 

0.10 

0.00 

0.00 

2.81 

3.77 

3.60 

8.46 

4.79 

2.75 

0.00 

0.00 

0.00 

0.00 

1.50 

0.00 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

Vehicle control for restart groups 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 

Study Design B 

Overview of the Sensitivity of Male SENCAR Mice in the Comparative InitiatiodPromotion Skin Paint Studies: 

TABLE C2b 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors (week) Total Micea Mice with Tumorsb 

100 pg MNNG/100 pg MNNG 

1000 pg MNNGDO mg BPO 

500 pg MNNGDO mg BPO 

100 pg MNNGfZO mg BPO 

1000 pg MNNGLI pg TPA 

100 pg MNNGD pg TPA 

1000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

20 mg BPOnO mg BPO 

1 pg TPN1 pg TPA 

Acetone/AcetoneC 

Acetone/Acetone 

29t30 

25/30 

25/30 

9/30 

2/30 

16/30 

22/30 

19/30 

u30 

OB0 

u30 

on0 

OB0 - 

- 

16.0 

29.4 

11.9 

12.9 

40.6 

11.8 

rn.8 

12.0 

13.9 

475 

- 

2.00 

1.70 

1.77 

0.50 

2.67 

1.47 

1.57 

1.37 

0.07 

0.00 

0.10 

0.00 

0.00 

2.07 

2.04 

2.12 

1.67 

3.33 

2.75 

2.14 

2.16 

1 .00 

0.00 

1.50 

0.00 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 

' Vehicle control for restart groups 
Lesions in Male and Female SENCAR Mice 155 

Study Design A 

Overview of the Sensitivity of Female SENCAR Mice in the Comparative Initiation/Promotion Skin Paint Studies: 

TABLE C2c 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors Total Micea Mice with Tumorsb (Week) 

2.5 pg DMBAL2.5 pg DMBA 

25 pg DMBArZ.0 mg BPO 

2.5 pg DMBm mg BPO 

25 kg DMBM pg TPA 

2.5 pg DMBAn kg TPA 

0.25 pg DMBAn pg "PA 

25 fig DMBNAcetone 

2.5 pg DMBNAcetone 

0.25 pg DMBNAcetone 

20 mg BPODO mg BPO 

1 pg TPAn fig PA 

Acetone/AcetoneC 

AcetondAcetone 

34.9 

33.6 

31.2 

14.4 

15.9 

23.5 

- 

2.80 

2.50 

2.33 

9.23 

5.23 

0.87 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

2.80 

3.75 

3.18 

9.55 

5.81 

2.00 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

Vehicle control for restart groups PUP. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 

Study Design B 

Overview of the Sensitivity of Female SENCAR Mice in the Comparative InitiatioWromotion Skin Paint Studies: 

TAELE C2d 

Mean Time Average Number Average Number 

Number of Mice to Tumor of Tumors/ of Tumors/ 

With Tumors Total Micea Mice with Tumorsb (Week) 

100 pg MNNGnOO pg MNNG 

1000 pg MNNGBO mg BPO 

500 pg MNNGEO mg BPO 

100 pg MNNGBO mg BPO 

1000 pg MNNG/l pg TPA 

100 pg MNNG/1 pg TPA 

1000 pg MNNG/Acetone 

500 pg MNNG/Acetone 

100 pg MNNG/Acetone 

20 mg BPOnO mg BPO 

1 pg TPA/1 kg TPA 

Acetone/Acetone' 

AcetonelAcetone 

28/30 

21/30 

16/30 

9/30 

20/30 

23/30 

25/30 

8/30 

on0 

1/30 

on0 

or30 

on0 

32.3 

12.5 

25.8 

38.1 

9.6 

23.0 

14.0 

25.5 

- 

1.77 

1.50 

0.73 

0.60 

1.93 

2.13 

1.87 

0.06 

0.00 

0.03 

0.00 

0.00 

0.00 

1.89 

2.14 

1.38 

2.00 

2.90 

2.78 

2.24 

2.25 

0.00 

1 .00 

0.00 

0.00 

0.00 

a Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of animals per group. 

Vehicle control for restart groups 

group. 

Maximum number of tumors recorded in-life per mouse were summed and divided by the total number of mice that developed tumors in each 
156 

~ 

Skin Paint Studies, NTP TR 441 

Initiatioflromotion Skin Paint Studies: Study Design Aa 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male SENCAR Mice in the Comparative 

TABLE C3a 

Acetonel 0.25 pg DMBY 2.5 pg DMBY 2.5 pg DMBY 

Acetone Acetone Acetone 2.5 pg DMBA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

SuMvors 

30 

1 

1 

30 

2 

1 

29 

1 

1 

30 

4 

15 

2.8 27 27 11 

Animals examined microscopically 30 30 29 30 

Hair follicle, site of 

skin 

Site of application-no mass, 

Site of application-no mass, acanthosis 

application-no mass, cyst 

Site of application-mass, cyst 

Site of application-mass, acanthosis 

Site of application-no mass, ulcer, multiple 

Site of application-no mass, ulcer 

inflammation, chronic active 

(30) (30) (29) (30) 

1 (3%) 

1 (3%) 

1 (3%) 4 (13%) 1 (3%) 

4 (13%) 

18 (60%) 

23 (77%) 

1 (3%) 

2 (7%) 

1 (3%) 

2 (7%) 

epithelial inclusion 
Lesions in Male and Female SENCAR Mice 157 

InitiatiodPromotion Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male SENCAR Mice in the Comparative 

TABLE C3a 

2.5 pg DMBN 25 pg DMBN 25 pg DMBN 20 mg BPO/ 

20 mg BPO Acetone 20 mg BPO 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

23 

1 

6 

30 

survivors 

Animals examined microscopically 30 

31 

31 

30 

1 

30 

1 

12 

17 

30 

30 

2 

5 

23 

30 

Hair follicle, site of 

Skin (continued) 

Site of application-no mass, 

Site of application-no mass, acanthosis 29 (97%) 

application-no mass, cyst 

Site of appliqtion-mass, cyst 

Site of application-mass, acanthosis 

Site of application-no mass, ulcer, multiple 

Site of application-no mass, ulcer 

inflammation, chronic active 29 (97%) 

(30) (31) (30) (30) 

3 (10%) 

28 (93%) 

28 (93%) 

1 (3%) 

1 (3%) 

29 (97%) 

30 (100%) 

2 (7%) 

1 (3%) 

4 (13%) 

epithelial inclusion 1 (3%) 

a Number of animals examined microscopically at site and number of animals with lesion 
158 Skin Paint Studies, NTP TR 441 

Initiatioflromotion Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male SENCAR Mice in the Comparative 

TABLE C3b 

Acetonel 100 pg MNNGI 500 pg MNNGI 1,000 pg MNNG/ 100 Pg mNG/ 

Acetone Acetone Acetone Acetone 100 pg MNNG 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Died last week of study 

Natural deaths 

Moribund 

30 

1 

Sulvivors 

29 

Animals exmined microscopically 30 

2 

30 

30 

28 

30 

4 

10 

30 

16 

30 

3 

20 

30 

7 

30 

3 

23 

30 

4 

Site of application-no mass, alopecia 

Site of application-no mass, acanthosis 1 (3%) 

Hair follicle, site of 

Skin (30) (30) (30) (30) (30) 

application-no mass, cyst 2 (7%) 2 (7%) 

6 (20%) 13 (43%) 9 (30%) 25 (83%) 

Site of application-no mass, 

Subcutaneous tissue, site of 

Site of application-mass, acanthosis 

Site of application-no mass, ulcer 

inflammation, chronic active 

Subcutaneous tissue, site of 

cellular, mixed cell 

application-no mass, infiltration 

application-no mass, edema 

2 (7%) 

8 (27%) 

15 (50%) 17 (57%) 

15 (50%) 

26 (87%) 

2 (7%) 

11 (37%) 

1 (3%) 
Lesions in Male and Female SENCAR Mice 159 

InitiationLPromotion Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male SENCAR Mice in the Comparative 

TABLE C3b 

20 mg BPO/ 100 kg MNNG/ 500 pg MNNG/ 1,000 kg MNNG/ 

20 mg BPO 20 mg BPO 20 mg BPO 20 mg BPO 

Early deaths 

Aniials initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Died last week of study 

Natural deaths 

Moribund 3 

30 

survivors 

26 

1 

Animals examined microscopically 30 

2 

30 

30 

28 

30 

3 

11 

16 

30 

30 

3 

21 

6 

30 

Hair follicle, site of 

Skin (continued) 

Site of application-no mass, 

Site of application-no mass, alopecia 

Site of application-no mass, acanthosis 

Subcutaneous tissue, site of 

Site of application-mass, acanthosis 

Site of application-no mass, ulcer 

inflammation, chronic active 

Subcutaneous tissue, site of 

application-no mass, cyst 

cellular, mixed cell 

application-no mass, infiltration 

application-no mass, edema 

(30) (30) (30) (30) 

28 (93%) 

1 (3%) 2 (7%) 

2.5 (83%) 27 (90%) 28 (93%) 

1 (3%) 

26 (87%) 

1 (3%) 3 (10%) 

1 (3%) 

29 (97%) 28 (93%) 

1 (3%) 

5 (17%) 

27 (90%) 

2 (7%) 5 (17%) 

~ ~ ~ ~~ ~ -~ ~ 

a Number of animals examined microscopically at site and number of animals with lesion 
160 Skin Paint Studies, NTP TR 441 

InitiatiodPromotion Skin Paint Studies: Study Restarta 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male SENCAR Mice in the Comparative 

TABLE C3c 

Acetonel 0.25 pg DMBN 2.5 pg DMBN 25 pg DMBN 

Acetone 1 fig TPA 1 fig TPA 1 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

SuMvors 

Animals examined microscopically 

1 

30 

30 

29 

30 

2 

22 

6 

30 

30 

6 

22 

2 

30 

30 

4 

25 

30 

1 

Hair follicle, site of 

Skin 

Site of application-no mass, 

Site of application-no mass, alopecia 

Site of application-no mass, acanthosis 

Site of application-mass, ulcer 

Site of application-mass, acanthosis 

Site of application-no mass, ulcer, multiple 

Site of application-no mass, ulcer 

inflammation, chronic active 

(30) (30) (30) (30) 

application-no mass, cyst 

30 (100%) 

5 (17%) 

30 (100%) 

6 (20%) 

29 (97%) 

9 (30%) 

2 (7%) 

2 (7%) 

12 (40%) 

9 (30%) 

30 (100%) 

9 (30%) 

14 (47%) 

30 (100%) 

15 (50%) 

28 (93%) 

6 (20%) 
Lesions in Male and Female SENCAR Mice 161 

Initiatioflromotion Skin Paint Studies: Study Restart ‘(continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Male SENCAR Mice in the Comparative 

TABLE C3c 

1 sg TPN 100 fig MNNG/ 

1 pg TPA 1 pg TPA 1 pg TPA 

1,000 pg MNNG/ 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

Survivors 

Animals examined microscopically 

30 

4 

17 

9 

30 

30 

2 

20 

8 

30 

30 

3 

26 

1 

30 

Hair follicle, site of 

Skin (continued) 

Site of application-no mass, 

Site of application-no mass, alopecia 

Site of application-no mass, acanthosis 

Site of application-mass, ulcer 

Site of applicatbn-mass, acanthosis 

Site of application-no mass, ulcer, multiple 

Site of application-no mass, ulcer 

inflammation, chronic active 

application-no mass, cyst 

30 (100%) 

15 (50%) 10 (33%) 

23 (77%) 

13 (43%) 

5 (17%) 

30 (100%) 

21 (70%) 

29 (97%) 

1 (3%) 

1 (3%) 

1 (3%) 

3 (10%) 

21 (70%) 

21 (70%) 

29 (97%) 

a Number of animals examined microscopically at site and number of animals with lesion 
162 

InitiatiodPromotion Skin Paint Studies: Study Design A" 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female SENCAR Mice in the Comparative 

TABLE C3d 

Acetone/ 0.25 pg DMBN 2.5 pg DMBN 2.5 fig DMBAY 

Acetone Acetone ketone 2.5 ,ug DMBA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

survivors 

Animals examined microscopically 

30 

1 

2 

27 

30 

1 

30 

30 

29 

1 

31 

31 

30 

30 

1 

19 

10 

30 

Site of application-no mass, 

Site of application-no mass, acanthosis 

Skin 

Site of application-no mass, ulcer, multiple 

Site of application-no mass, ulcer 

inflammation, chronic active 

(29) (30) (31) (30) 

2 (7%) 

2 (7%) 3 (10%) 23 (77%) 

1 (3%) 

3 (10%) 

2 (7% 5 (16%) 22 (73%) 

1 (3%) 
Lesions in Male and Female SENCAR Mice 163 

InitiatiodPromotion Skin Paint Studies: Study Design A (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female SENCAR Mice in the Comparative 

TABLE C3d 

2.5 pg DMBA/ 25 pg DMBA/ 25 pg DMBA/ 20 mg BPO/ 

20 mg BPO Acetone 20 mg BPO 20 mg BPO 

Animals examined microscopically 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

Sulvivors 

30 

1 

8 

21 

30 

29 

1 

29 

28 

30 

1 

6 

23 

30 

2 

30 

30 

28 

Site of application-no mass, 

Site of application-no mass, acanthosis 25 (83%) 

Skin (continued) 

Site of application-no mass, ulcer, multiple 

Site of application-no mass, ulcer 

inflammation, chronic active 30 (100%) 

(30) (29) (30) (30) 

3 (10%) 

3 (10%) 

27 (90%) 

27 (90%) 16 (53%) 

28 (93%) 

1 (3%) 

3 (10%) 

1 (3%) 

1 (3%) 

a, Number of animals examined microscopically at site and number of animals with lesion 
164 Skin Paint Studies, NTP TR 441 

Initiatioflromotion Skin Paint Studies: Study Design Ba 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female SENCAR Mice in the Comparative 

TABLE C3e 

Acetone/ 100 pg MNNG/ 500 pg MNNG/ 1,000 pg MNNG/ 100 pg MNNG/ 

Acetone Acetone Acetone Acetone 100 pg MNNG 

Early deaths 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

SuMvors 

30 

2 

1 

1 

26 

30 

30 

30 

3 

5 

22 

30 

6 

13 

11 

30 

2 

25 

3 

Animals exmined microscopically 30 30 30 30 30 

Hair follicle, site of 

Skin 

Site of application-no mass, 

Site of application-no mass, acanthosis 

Site of application-no mass, abscess 

Site of application-mass, acanthosis 

Site of application-no mass, ulcer 

inflammation, chronic active 

(30) 

application-no mass, cyst 

11 (37%) 

2 (7%) 

1 (3%) 

1 (3%) 

16 (53%) 

17 (57%) 

6 (20%) 

27 (90%) 

24 (80%) 

8 (27%) 

16 (53%) 

1 (3%) 

2 (7%) 
Lesions in Male and Female SENCAR Mice 165 

Initiatioflromotion Skin Paint Studies: Study Design B (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female SENCAR Mice in the Comparative 

TABLE C3e 

20 mg BPO/ 100 pg MNNG/ 500 pg MNNGl 1,000 kg MNNG/ 

20 mg BPO 20 mg BPO 20 mg BPO 20 mg BPO 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

30 

27 

2 

1 

Survivors 

Animals examined microscopically 30 

30 

1 

30 

29 

30 

7 

30 

23 

30 

4 

12 

14 

30 

Hair follicle, site of 

Skin (continued) 

Site of application-no mass, 

Site of application-no mass, acanthosis 

Site of application-no mass, abscess 

Site of application-mass, acanthosis 

Site of application-no mass, ulcer 

inflammation, chronic active 

(30) (30) (30) (30) 

application-no mass, cyst 1 (3%) 

27 (90%) 

27 (90%) 28 (93%) 28 (93%) 

1 (3%) 

2 (7%) 

28 (93%) 28 (93%) 

6 (20%) 

29 (97%) 

27 (90%) 

1 (3%) 

~~~~ ~ ~ ~ ~~ ~~~ 

a Number of animals examined microscopically at site and number of animals with lesion 
166 Skin Paint Studies, NTP TR 441 

InitiatiodPromotion Skin Paint Studies: Study Restarta 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female SENCAR Mice in the Comparative 

TABLE C3f 

Acetone 

Acetonel 0.25 pg DMBN 2.5 pg DMBA/ 25 pg DMBN 

1 Kg TPA 1 pg TPA 1 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

Survivors 

30 

2 

1 

27 

30 

4 

18 

8 

30 

5 

19 

6 

30 

6 

23 

1 

Animals examined microscopically 30 30 30 30 

Site of application-no mass, acanthosis 

Hair follicle, site of 

Skin (29) 

application-no mass, cyst 

(30) (30) (30) 

30 (100%) 

12 (40%) 

30 (100%) 

8 (27%) 9 (30%) 

28 (93%) 

Site of application-no mass, 

Site of application-mass, cyst 

Site of application-mass, acanthosis 

Site of application-no mass, ulcer, multiple 

Site of application-no mass, ulcer 

inflammation, chronic active 

1 (3%) 

1 (3%) 

12 (40%) 

8 (27%) 

30 (100%) 

6 (20%) 

30 (100%) 

9 (30%) 

13 (43%) 

27 (90%) 

1 (3%) 

15 (50%) 

1 (3%) 

epithelial inclusion 
Lesions in Male and Female SENCAR Mice 167 

Initiatioflromotion Skin Paint Studies: Study Restart (continued) 

Summary of the Incidence of Nonneoplastic Skin Lesions in Female SENCAR Mice in the Comparative 

TABLE C3f 

1 ccg TPN 100 fig MNNG/ 1,000 fig MNNG/ 

1 pg TPA 1 pg TPA 1 pg TPA 

Early deaths 

Animals initially in study 

Disposition Summary (continued) 

Terminal sacrifice 

Natural deaths 

Moribund 

SuMvors 

Animals examined microscopically 

30 

1 

19 

10 

30 

30 

2 

15 

13 

30 

30 

4 

24 

2 

30 

Hair follicle, site of 

Skin (continued) 

Site of application-no mass, 

Site of application-no mass, acanthosis 

application-no mass, cyst 

Site of application-mass, cyst 

Site of application-mass, acanthosis 

Site of application-no mass, ulcer, multiple 

Site of application-no mass, ulcer 

inflammation, chronic active 

epithelial inclusion 

5 (17%) 

30 (100%) 

30 (100%) 

16 (53%) 

24 (80%) 

5 (17%) 

28 (93%) 

19 (63%) 

1 (3%) 

11 (37%) 

1 (3%) 

(30) 

3 (10%) 

17 (57%) 

23 (77%) 

24 (80%) 

1 (3%) 

2 (7%) 

a Number of animals examined microscopically at site and number of animals with lesion 
168 Skin Paint Studies, NTP TR 441 
169 

DOSE FORMULATION STUDIES 

CHEMICAL CHARACTERIZATION AND 

APPENDIX D 

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS 

PROCUREMENT AND CHARACTERIZATION .............................................. 172 

170 

.................................... 

TABLE Dl 

FIGURE D7 

FIGURE D6 

FIGURE D5 

FIGURE D4 

FIGURE D3 

FIGURE D2 

FIGURE Dl 

TABLE D2 

TABLE D3 

Skin Paint Studies 

Swiss (CD-1") Mice. and SENCAR Mice in the Comparative InitiatiodPromotion 

Results of Referee Analysis of Dose Formulations Administered to B6C3Fl Mice. 

Skin Paint Studies 

Swiss (CD-1") Mice. and SENCAR Mice in the Comparative Initiatioflromotion 

Results of Analysis of Dose Formulations Administered to B6C3Fl Mice. 

in the Comparative InitiationlPromotion Skin Paint Studies 182 

Preparation and Storage of Dose Formulations 

Nuclear Magnetic Resonance Spectrum of 12-0-Tetradecanoylphorbol-13-acetate 181 

Nuclear Magnetic Resonance Spectrum of N-Methyl-N'-nitro-N-nitrosoguanidine 180 

Infrared Absorption Spectrum of N-Methyl-N'-nitro-N-nitrosoguanidine 179 

Nuclear Magnetic Resonance Spectrum of 7.1 2.Dimethylbenz(a)anthracene 178 

Infrared Absorption Spectrum of 7.1 2.Dimethylbenz(a)anthracene 177 

Nuclear Magnetic Resonance Spectrum of Benzoyl Peroxide 176 

Infrared Absorption Spectrum of Benzoyl Peroxide ........................... 175 

.................... ................ ......... ............ ..... 

..... 

.................... 

.................................................... 183 

.................................................... 186 
170 Skin Paint Studies, NTP TR 441 

DOSE FORMULATION STUDIES 

CHEMICAL CHARACTERIZATION AND 

Institute of Environmental Health Sciences (NIEHS). 

laboratory. Reports on analyses performed in support of the 1-year studies are on file at the National 

chemistry laboratory, Midwest Research Institute (Kansas City, MO) and confirmed by the study 

was used throughout the 1-year studies. Identity and purity analyses were conducted by the analytical 

Benzoyl peroxide was obtained from Akzo Chemie America (Maple Shade, NJ) in one lot (WM-40) which 

Benzoyl Peroxide 

PROCUREMENT AND CHARACTERIZATION 

(Sudtler Standard Spectra) of benzoyl peroxide (Figures Dl and D2). 

and nuclear magnetic resonance spectroscopy. All spectra were consistent with the literature spectra 

The chemical, a white powdered solid, was identified as benzoyl peroxide by infrared, ultraviolet/visible, 

1.0 mL/minute, detection at 254 nm, and a solvent system of water:acetonitrile (40:60). 

thiocyanate. HPLC was performed with a Waters pBondapak C,, column, with a flow rate of 

a reference standard. Plates were examined under 254 nm ultraviolet light and a spray of ferrous 

tetrach1oride:glacial acetic acid (66:33:1), and 2) ch1oroform:ether (7525). Benzoic anhydride was used as 

endpoint. TLC was performed on Silica Gel 60 F-254 plates with two solvent systems: 1) to1uene:carbon 

solution (1:2), and immediately titrating the samples with 0.1 N sodium thiosulfate to a colorimetric 

Functional group titration was performed by dissolving samples in acetone, adding potassium:iodide 

titration, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). 

Purity was determined by elemental analyses, nuclear magnetic resonance spectroscopy, functional group 

determined to be approximately 99% when corrected for water content. 

indicated two impurities with peak areas totaling 0.4% relative to the major peak. The overall purity was 

97% +- 2(s)% calculated on an anhydrous basis. TLC indicated a major spot by each system. HPLC 

spectroscopy indicated 19.7% f 0.5(s)% water. Functional group titration indicated a purity of 

Elemental analyses for carbon and hydrogen agreed with theoretical values. Nuclear magnetic resonance 

laboratory using HPLC and ultraviolet spectroscopy. No degradation of the bulk chemical was observed. 

refrigeration. During the 1-year studies, the stability of the bulk chemical was monitored by the study 

bulk chemical be stored protected from light at 5" C. Bulk benzoyl peroxide was stored under 

Based on half-life data from the manufacturer the analytical chemistry laboratory recommended that the 

1-year studies are on file at the NIEHS. 

identity, purity, and stability analyses performed by the analytical chemistry laboratory in support of the 

material was assigned lot number M111384 and was used throughout the 1-year studies. Reports on the 

benzene, passed through a neutral alumina column, and crystallized from isopropanol. The purified 

lot (K-4). The lot was purified by the analytical chemistry laboratory. The chemical was dissolved in 

7,12-Dimethylbenz(a)anthracene was obtained from the Eastman Kodak Company (Rochester, NY) in one 

7,12-Dimethylbenz(a)anthracene 

literature spectra (SudtZer Standard Spectra) of 7,12-dimethylbenz(a)anthracene (Figures D3 and D4). 

ultraviolet/visible, and nuclear magnetic resonance spectroscopy. All spectra were consistent with the 

The chemical, a light yellow powder, was identified as 7,12-dimethylbenz(a)anthracene by infrared, 

chromatography. TLC was performed on Silica Gel 60 F-254 plates using two solvent systems: 

The purity was determined by elemental analyses, Karl Fischer water analysis, TLC, and gas 
Chemical Characterization and Dose Formulation Studies 171 

ionization detector with a nitrogen carrier gas at a flow rate of 70 mL/minute. Two systems were used: 

of 5% potassium dichromate in 40% sulfuric acid. Gas chromatography was performed using a flame 

used as a reference standard. Plates were examined under 254 nm and 366 nm ultraviolet light and a spray 

1) to1uene:hexane (60:40) and 2) hexane:chloroform (7822). Pyrene dissolved in toluene (1OpuglmL) was 

A) 3% Dexsil400 on SO/lOO Chromosorb W(AW) column, with an oven temperature program of 

50" C for 5 minutes, then 50" to 300" C at 10" C per minute, and 

B) 3% SP-2100 on 100/120 Supelcoport column, with an oven temperature program of 75" C for 

1 minute, then 75" to 275" C at 10" C per minute. 

0.1% relative to the major peak area. The overall purity was determined to be greater than 99%. 

chromatography using both systems indicated one major peak and no impurities with peaks greater than 

system 1 indicated one major spot and one trace spot, and system 2 indicated only a major spot. Gas 

7,12-dimethylbenz(a)anthracene. Karl Fischer water analysis indicated less than 0.4% water. TLC by 

Elemental analyses for carbon and hydrogen were in agreement with the theoretical values for 

degradation of the bulk chemical was observed. 

periodically by the study laboratory using ultraviolet spectroscopy and gas chromatography. No 

protected from light at approximately -20" C. The stability of the bnlk chemical was monitored 

protected from light at temperatures up to 60" C. To ensure stability, the bulk chemical was stored 

that 7,12-dimethylbenz(a)anthracene was stable as a bulk chemical for at least 2 weeks when stored 

temperature of 300" C and 2.3 muglmL octacosane added as an internal standard. These studies indicated 

Stability studies were performed with gas chromatography system A described above except .with an oven 

analyses performed in support of the 1-year studies are on file at the NIEHS. 

conducted by the analytical chemistry laboratory and confirmed by the study laboratory. Reports on 

one lot (8228CK) which was used throughout the 1-year studies. Identity and purity analyses were 

N-Methyl-N'-nitro-N-nitrosoguanidine was obtained from Aldrich Chemical Company (Milwaukee, WI) in 

N-Methyl-N'-nitro-N-nitrosoguanidine 

and D6). 

the literature spectra (Sadtler Standard Spectra) of N-methyl-N'-nitro-N-nitrosoguanidine (Figures D5 

infrared, ultravioletMsible, and nuclear magnetic resonance spectroscopy. All spectra were consistent with 

The chemical, a light yellow crystalline solid, was identified as N-methyl-N'-nitro-N-nitrosoguanidine by 

hexane:methylene chloride (60:40). 

CN column, with a flow rate of 1.0 mL/minute, detection at 254 nm, and a solvent system of 

254 nm ultraviolet light, with a titaneous chloride spray. HPLC was performed with an Alltech Spherisorb 

2) to1uene:ethyl acetate (l:l), with p-nitroaniline as a reference standard. Plates were examined under 

TLC was performed on Silica Gel 60 F-254 plates using two solvent systems: 1) to1uene:acetone (9:1), and 

Purity was determined by elemental analyses, nuclear magnetic resonance spectroscopy, TLC, and HPLC. 

approximately 99%. 

areas greater than 0.1% relative to the major peak. The overall purity was determined to be 

with a minor, a trace, and a slight trace impurity by system 2. HPLC indicated no impurities with peak ' 

chemical was negligible. TLC indicated a major spot and a minor impurity by system 1 and a major spot 

magnetic resonance spectrum and elemental analysis results indicated that the water content of the 

Elemental analyses for carbon, hydrogen, and nitrogen agreed with theoretical values. The nuclear 

protected from light. The bulk chemical was stored at -20" C in amber glass bottles. The stability of the 

The manufacturer's information and literature sources specified that the bulk chemical be stored at -20" C 
172 Skin Paint Studies, NTP TR 441 

No degradation of the bulk chemical was observed. 

bulk chemical was monitored periodically by the study laboratory using HPLC and ultraviolet spectroscopy. 

of the study are on file at the NIEHS. 

analyses were conducted by the analytical chemistry laboratory. Reports on analyses performed in support 

to assist in tracking. All five lots were used during the 1-year studies. Identity, purity, and stability 

of lot 00411999 was received from Pharmacia PL Biochemical and was assigned a new number (UN2811) 

and OE11999), and from L.C. Services Corporation (Woburn, MA) in one lot (F-121). A second shipment 

(Brewster, NY) in one lot (031), from Pharmacia PL Biochemical (Milwaukee, WI) in two lots (00411999 

12-0-Tetradecanoylphorbol-13-acetate was obtained from Consolidated Midland Corporation 

12-0-Tetradecanoylphorbol-13-acetate 

12-0-tetradecanoylphorbol-13-acetate (Figure D7). 

resonance spectroscopy and mass spectrometry. Both spectra were consistent with that expected for 

Each lot of the chemical was identified as 12-0-tetradecanoylphorbol-13-acetate by nuclear magnetic 

system of water:acetonitrile (10:90). 

a DuPont Zorbax ODS column, with a flow rate of 1 mL/minute, detection at 229 nm, and a solvent 

concentrated sulfuric acid, followed by heating at 120" C for 10 to 20 minutes. HPLC was performed with 

(60:40). Visualization was at 254 nm (and 366 nm for lot 00411999) with a spray of 1% vanillin in 

plates using two solvent systems: 1) anhydrous diethyl ether (loo%), and 2) ethyl acetate:chloroform 

The purity of the five lots was determined by TLC and HPLC. TLC was performed on Silica Gel 60 F-254 

UN2811. 

purity was determined to be 99% for lots F-121, 0E11999, and 00411999 and 97% for lots 031 and 

to 0.1% of the major peak area and a combined area of 0.8% relative to the major peak area. The overall 

area. For lot F-121, HPLC indicated one major peak and two impurities with areas greater than or equal 

than or equal to 0.1% of the major peak area and a combined area of 1.0% relative to the major peak 

the major peak. HPLC of lot OB11999 indicated one major peak and five impurities with areas greater 

with areas greater than or equal to 0.1% of the major peak area and a combined area of 0.6% relative to 

relative to the major peak area. For lot 00411999, HPLC indicated one major peak and three impurities 

impurities with areas greater than or equal to 0.1% of the major peak area and a combined area of 2.9% 

3.1% relative to the major peak area. HPLC of lot UN2811 indicated one major peak and seven 

11 impurities with areas greater than or equal to 0.1% of the major peak area and a combined area of 

impurity, and two very slight trace impurities. HPLC of lot 031 revealed one major peak and 

one very slight trace impurity, while system 2 revealed one major spot, one trace impurity, one slight trace 

impurity using system 2. TLC of lot 031 using system 1 revealed one major spot, one trace impurity, and 

lot 00411999 revealed only one major spot using system 1 and one major spot and one very slight trace 

TLC for lots UN2811, 0E11999, and F-121 revealed one major spot with each system. TLC for 

temperature for up to 6 days. 

study indicated that no decomposition had occurred in samples exposed to air and light at ambient 

The stability of the chemical was determined using the HPLC system described in the purity analysis. The 

from light, and were discarded 3 weeks after preparation. 

concentrations (Table Dl). The dose formulations were stored at 4" C or at room temperature protected 

Dose formulations were prepared by dissolving benzoyl peroxide in acetone to give the required 

Benzoyl Peroxide 

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS 
Chemical Characterization and Dose Formulation Studies 173 

study samples were prepared by dissolving benzoyl peroxide in acetone and sonicating. 

Dose formulation stability studies were performed by the analytical chemistry laboratory. The stability 

sealed vials in the dark at 5" C, and for 3 hours when the chemical was exposed to light and air. 

solutions in acetone (100 mg/mL) was confirmed for at least 3 weeks when the solutions were stored in 

(75:25), with benzoyl peroxide 64 pg/mL, and detection at 280 nm. The stability of benzoyl peroxide 

Waters pBondapak C18column, with a flow rate of 1 mL/minute, a mobile phase of acetonitri1e:water 

(0.5 mg/mL in acetonitri1e:water [75:25]) then filtered (0.45 p pore size) and analyzed by HPLC using a 

diluted to volume with acetone. Aliquots of diluted samples were combined with a phenanthrene solution 

from the 3-hour study were brought to their original weight by adding acetone, and aliquots (4 mL) were 

Samples to be analyzed from the 21-day stability study were first diluted to volume with acetone. Samples 

formulations (Table D3). 

laboratory were in agreement with the results obtained by the study laboratory for two of the four 

target concentration. Results of the periodic referee analyses performed by the analytical chemistry 

(Table D2). During the 1-year studies, 94% (15/16) of the dose formulations were within 10% of the 

analytical chemistry laboratory using HPLC. The dose formulations were analyzed at least every 8 weeks 

Periodic analysis of dose formulations of benzoyl peroxide were performed by the study laboratory and the 

discarded 3 weeks after preparation. 

required concentration (Table Dl). The dose formulations were stored at 4" C protected from light, and 

The dose formulations were prepared by dissolving 7,12-dimethylbenz(a)anthracene and acetone to give the 

7,12-Dimethylbenz(a)anthracene 

when stored in the dark, and for less than 3 hours when exposed to light and air. 

365 nm. The stability of the dose formulations was confirmed for at least 3 weeks at room temperature 

mobile phase of acetonitri1e:water (85:15), with anthracene added as an internal standard, and detection at 

[85:15]). HPLC was performed using a Brownlee RP-18 column, with a flow rate of 1 mL/minute, a 

sslution, anthracene (0.2 mg/mL in acetonitri1e:water [85:15], and further diluted with acetonitri1e:water 

chemistry laboratory. Aliquots were diluted with acetone, then mixed with 5 mL of the internal standard 

Stability analyses of the 0.1 mg/mL and 0.0025 mg/mL dose formulations were performed by the analytical 

with the results obtained by the study laboratory (Table D3). 

Results of periodic referee analyses performed by the analytical chemistry laboratory were in agreement 

studies, 100% (23/23) of the dose formulations were within 10% of the target concentrations (Table D2). 

laboratory and analytical chemistry laboratory using ultraviolet spectroscopy at 363 nm. During the 1-year 

Periodic analyses of the dose formulations of 7,1Zdimethylbenz(a)anthracene were conducted by the'study 

and were discarded 3 weeks after preparation. 

the required concentrations (Table Dl). The dose formulations were stored at 4" C protected from light, 

Dose formulations were prepared by dissolving N-methyl-N'-nitro-N-nitrosoguanidine in acetone to give 

N-Methyl-N'-nitro-N-nitrosoguanidine 

exposed to light and air. 

confirmed for at least 3 weeks in sealed vials, in the dark, at room temperature, and for 3 hours when 

an internal standard, and detection at 280 nm. The stability of N-methyl-N'-nitro-N-nitrosoguanidine was 

n-hexane:methylene chloride (50:50), with N-methyl-N'-nitro-N-nitrosoguanidine (10.1 pg/mL) added as 

Alltech Associates Spherisorb S-5-CN column, with a flow rate of 1 mL/minute, a mobile phase of 

chloride) and diluted to 100 mL with n-hexane:methylene chloride (5050). HPLC was performed using an 

Aliquots (2 mL) of these solutions were mixed with 3 mL of dimethylnitrosamine (45 mg/mL in methylene 

prepared by dissolving 50 mg of N-methyl-N'-nitro-N-nitrosoguanidine in acetone and diluting to 100 mL. 

Dose formulation stability studies were performed by the analytical chemistry laboratory. Samples were 
174 Skin Paint Studies, NTP TR 441 

for two of the three formulations. 

performed by the analytical laboratory were in agreement with the results obtained by the study laboratory 

formulations were within 10% of the target concentration. Results of the periodic referee analyses 

analyzed at least every 8 weeks (Table D2). During the 1-year studies, 95% (19DO) of the dose 

study laboratory and the analytical chemistry laboratory using HPLC. The dose formulations were 

Periodic analyses of dose formulations of N-methyl-N'-nitro-N-nitrosoguanidine were performed by the 

preparation. 

formulations were stored at 4" C protected from light, and were discarded 3 weeks after the date of 

the required concentrations (Table Dl). Dose formulations were prepared every 2 weeks. The dose 

The dose formulations were prepared by mixing 12-0-tetradecanoylphorbol-13-acetate and acetone to give 

12-0-Tetradecanoylphorbol-13-acetate 

temperature in amber glass bottles. 

solvent ratio of 7:93. Stability of the formulation was established for at least 3 weeks when stored at room 

HPLC system used in the bulk chemical analyses of 12-0-tetradecanoylphorbol-13-acetate except with a 

Stability analyses of the dose formulations were conducted by the analytical chemistry laboratory, using the 

formulations (Table D3). 

chemistry laboratory were in agreement with the results obtained by the study laboratory in three of the six 

of the target concentrations (Table D2). Results of periodic referee analyses performed by the analytical 

the stability study. During the 1-year studies, 92% (24D6) of the formulations analyzed were within 10% 

study laboratory and by the analytical chemistry laboratory with the same HPLC method as that used in 

Periodic analyses of the dose formulations of 12-0-tetradecanoylphorbol-13-acetate were conducted by the 
Chemical Characterization and Dose Formulation Studies 

0 

(D 

175 

Infrared Absorption Spectrum of Benzoyl Peroxide 

FIGURE Dl 
176 Skin Paint Studies, NTP TR 441 

w 

t 

! 

dd 

I T* 

:f 

I 

Nuclear Magnetic Resonance Spectrum of Benzoyl Peroxide 

FIGURE D2 

ksl nmenlr 

3.16 3 

'WL Oblewed Theoretical 

Integration 

(a) 3.00 

(b) 3.29 3.08 3 

5.29 5 

0.82 1 

J,-f = 9Hx 0.76 1 

(h) 8.41 - 8.48 

jj[[26 - 8.37 

(d) 7.76 - 7.82 

(c) 7.46 - 7.67 

] 1.97 

2 

0.92 I 

(I) 7.36 

(k) Solvent, 7.20 

(i) 1.48 

(I) 1.19 

Impurities: 

trace 

trace - 

- - - 

0.08 .. 

- 

Project No.: 8402-05 

MRI Task No.: BS/CV-1414 

Botch No, : 03 

Lot No. : M I1 1384 

297N 7.12-Dimc~hylbenrla]on~hracene 

Somple Temperalure: Ambient 

Zero Reference: TMS 

Nucleus: Proton 

Solvent: Deuterated Chloroform 

Instrument: Jeol 300- MHz FT- NMR 

Oprotor: 0. Taylor 

Doter 12/20/84 

I 

1 

v1 

B 

8 6 i 2 0 

PPm 
Chemical Characterization and Dose Formulation Studies 179 

Infrared Absorption Spectrum of N-Methyl-N'-nitro-N-nitrosoguanidine 

FIGURE D5 
A 

.fP 
ONE-PULSE SEOUENCE 

P2. 7.00 USEC 

D5- 10.00 SEC 

NA 32 

DW 150 

SY . .I- 1000.00 

AI = 4 

AM: = 12 BlTS 

08 ATT. * 

BUTTERYORTW FILTER ON 

A8C ON 

OPD OW l 

.. 

DE. 250 USEC 

RC . 10 USEC 

BB MODULATION ON 

F2. 300.054720 

TL IllGW POYER ON 

1 

3 

Qburrrp 

--- 

Ih.nr97.48 

2.97 

2.89 

I .90 

2.97 

5.95 

2.89 

0.99 

1.98 

0.99 

I .98 

I .98 

0.99 

0. 28 

I 

2 

I 

2 

2 

1 

1 

I 

2 

3 

7 

3 

22 

4 

3 

-- 
182 Skin Paint Studies, NTP TR 44% 

Skin Paint Studies 

Preparation and Storage of Dose Formulations in the Comparative Initiation/Promotion 

TABLE Dl 

benz(a)anthracene 

7,12-Dimethyl- N-Methyl-N'-nitro-N- 12-0-Tetradecanoylnitrosoguanidine phorbol-ffacetate Benzoyl Peroxide 

concentration. 

required benzoyl peroxide 

obtain a solution with the 

Acetone was added to' 

graduated cylinder. ' 

transferred to a 

weighed and then 

Benzoyl peroxide was. ). 

Preparation 

Chemical Lot Number 

WM-40 

3 weeks 

Maximum Storage Time 

temperature) 

1985 (stored at room 

prepared June 10-25, 

from light, except doses 

Stored at 4 "C protected 

Storage Conditions 

(Columbus, OH) 

Division 

Battelle Columbus 

Study Laboratory 

(Kansas City, MO) 

Institute 

Midwest Research 

Referee Laboratory 

concentration. 

benz(a)anthracene 

required 7J2-dimethylobtain a solution with the 

Acetone was added to 

graduated cylinder. 

transferred to a 

weighed and then 

(a)anthracene was 

7J2-DirnethylbenzM111384 

3 weeks 

from light 

Stored at 4 "C protected 

(Columbus, OH) 

Division 

Battelle Columbus 

(Kansas City, MO) 

Institute 

Midwest Research 

concentration. 

nitro-N-nitrosoguanidine 

required N-methyl-N'- 

obtain a solution with the 

Acetone was added to 

graduated cylinder. 

transferred to a 

weighed and then 

nitrosoguanidine was 

N-Methyl-N'-nitro-N8228CK 

3 weeks 

from light 

Stored at 4 "C protected 

(Columbus, OH) 

Division 

Battelle Columbus 

(Kansas City, MO) 

Institute 

Midwest Research 

concentration. 

phorbol-13-acetate 

12-0-tetrademnoylwith the required 

obtain a solution 

Acetone was added to 

graduated cylinder. 

transferred to a 

weighed and then 

phorbol-1Zacetate was 

12-0-TetradecanoylOEiE999, and F-121 

031, UN2811,00411999, 

3 weeks 

from light 

Stored at 4 "C protected 

(Columbus, OH) 

Division 

Battelle Columbus 

(Kansas City, MO) 

Institute 

Midwest Research 
Chmical Characterization and Dose Formulation Studies 183 

and SENCAR Mice in the Comparative Initiation/Promotion Skin Paint Studies 

Results of Analysis of Dose Formulations Administered to B6C3Fl Mice, Swiss (CD-la) Mice, 

TABLE D2 

Concentration 

Target Determined % Difference 

Date Prepared Date Analyzed Concentration from Target 

(m&wa (mglWb 

Benzoyl Peroxide 

18 April 1985 

1 May 1985' 

19 April 1985 

100.0 

100.0 

106.24 

96.04 

+6 

-4 

10 June 1985 14June1985 100.0 96.36 -4 

5 August 1985 

30 September 1985 2 October 1985 

5 September 1985' 

7 August 1985 

26 November 1985 26November 1985 

18 February 1986' 

21 January 1986 23 January 1986 

18 March 1986 20 March 1986 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

107.59 

98.67 

101.97 

97.41 

93.14 

94.50 

109.06 

115.56 

99.03 

101.17 

98.28 

97.20 

-1 

+a 

+2 

-3 

-7 

-6 

+9 

+ 16 

-1 

+1 

-2 

-3 

12 May 1986 14 May 1986 100.0 98.66 -1 

7,12-Dimethylbenz(a)anthracene 

18 April 1985 19 April 1985 

2 May 1985' 

0.500 

0.250 

0.025 

0.0025 

0.500 

0.250 

0.025 

0.0025 

0.0240d 

0.0023d 

0.49708 

0.26624 

0.02631d 

0.00236d 

0.4960 

0.2480 

-1 

+6 

+5 

-6 

-1 

-1 

-4 

-8 

5 August 1985 

11June1985 

1 May 1985 

6 August 1985 

13 June 1985 

2 May 1985 

0.025 

0.025 

0.025 0.0245ld 

+1 

+4 

-2 

0.02529d 

0.02593d 

24 October 1985' 

8 October 1985 10 October 1985 

24 October 1985' 

1 October 1985 3 October 1985 

0.025 

0.025 

0.025 

0.025 

0.02685 

0.02588d 

0.02648 

0.02609d 

+7 

+4 

t6 

+4 
184 Skin Paint Studies, NTP TR 441 

and SENCAR Mice in the Comparative Initiatioflromotion Skin Paint Studies (continued) 

Results of Analysis of Dose Formulations Administered to B6C3Fl Mice, Swiss (CD-lm) Mice, 

TABLE D2 

Target Determined % Difference 

Date Prepared Date Analyzed Concentration Concentration from Target 

(mg/mL) (mg/W 

7,12-Dimethylbenz(a)anthracene (continued) 

24 October 1985' 

11 October 1985 11 October 1985 

0.250 

0.0025 

0.250 

0.0025 0.00258d 

+2 

+5 

+1 

+3 

0.2551 

0.00262 

0.2522 

25 November 1985 2November 1985 0.025 0.02606 +4 

21 January 1986 23 January 1986 0.025 0.02607d +4 

10 April 1986' 

18 March 1986 18 March 1986 

N-Methyl-N'-nitro-N-nitrosoguanidine 

17 May 1985 20 May 1985 

3-4 June 1985' 

5 June 1985' 

0.025 

0.025 

10.0 

5.0 

10.0 

5.0 

1 .o 

10.0 

5.0 

1 .o 

0.02635d 

0.02589d 

+5 

+4 

10.69 

5.43 

12.71f 

5.42e 

1.10 

10.66 

5.46 

1.00 

+8 

+1 

+7 

+9 

0 

+7 

+9 

+ 27 

8 July 1985 9 July 1985 1.0 0.96 -4 

5 September 1985 6 September 1985 1 .o 1.01 +1 

21 October 1985' 

8 October 1985 15 October 1985 

18 October 1985' 

15 October 1985 15 October 1985 

19 November 1985' 

28 October 1985 31 October 1985 

10.0 

10.0 

1.0 

1 .o 

1 .o 

1 .o 

10.95 

10.34 

0.93 

0.94e 

1.07 

0.96 

+ 10 

+3 

-1 

-7 

+7 

-4 

23 December 1985 27December 1985 1 .o 0.95 -5 

18 February 1986 19February 1986 1 .o 0.94 -6 

14 April 1986 

7 May 1986' 

15 April 1986 

1.0 

1 .o 

0.96 

1.03 +3 .. 

-4 
Chemical Characterization and Dose Formulation Studies 185 

and SENCAR Mice in the Comparative InitiatiolllPromotion Skin Paint Studies (continued) 

Results of Analysis of Dose Formulations Administered to B6C3Fl Mice, Swiss (CD-la) Mice, 

TABLE D2 

Target Determined % Difference 

Date Prepared Date Analyzed Concentration Concentration from Target 

(mp/mL) (mglmL) 

18 April 1985 

30 September 1985 

7 August 1985 

5 August 1985 

10 June 1985 

1 May 1985 

8 October 1985 

25 November 1985 

21 January 1986 

18 March 1986 

12 May 1986 

2 September 1986 

7 July 1986 

3 May 1985 

3 May 1985' 

19 April 1985 

12 June 1985 

3 October 1985 

7 August 1985 

6 August 1985 

22 October 1985' 

22 October 1985' 

14 October 1985 

2 December 1985 

26 November 1985 

25 January 1986 

8 April 1986' 

21 March 1986 

19 March 1986 

4 June 1986' 

13 May 1986 

8 July 1986 

22 September 1986' 

3 September 1986 

0.05 

0.05 

0.05 

0.05 

0.05 

0.05 

0.05 

0.01 

0.05 

0.01 

0.05 

0.01 

0.01 

0.05 

0.01 

0.05 

0.05 

0.01 

0.05 

0.05 

0.01 

0.01 

0.05 

0.01 

0.01 

0.01 

0.0537 

0.05182 

0.05326 

0.05664 

0.0479 

0.0519 

0.0518 

0.01016 

0.04680 

0.00998 

0.04971 

0.00991 

0.00961 

0.04964 

0.01385 

0.05489 

0.05451 

0.01036 

0.04664 

0.04855 

0.00988 

0.00904 

0.04561 

0.01041e 

0.01006 

0.01013 

+7 

+4 

+7 

+13 

-4 

+4 

+4 

+2 

-6 

0 

-1 

-1 

-4 

-1 

+39 

+ 10 

+9 

+4 

-6 

-3 

-1 

-10 

-9 

+1 

+1 

+4 

a Dosing volume = 0.1 mL for 7,12-dimethylbenz(a)anthracene, N-methyl-N'-nitro-N-nitrosoguanidine, and 

Results of duplicate analyses 

0.1 mL) 

12-0-tetradecanoylphorbol-13-acetate. Dosing volume for benzoyl peroxide was 0.2 mL (two consecutive administrations of 

Results of quadruplicate analyses 

e Results of triplicate analyses 

Results of single analysis 

' Animal room sample 
186 Skin Paint Studies, NTP TR 441 

and SENCAR Mice in the Comparative InitiatiodPromotion Skin Paint Studies 

Results of Referee Analysis of Dose Formulations Administered to B6C3Fl Mice, Swiss (CD-la) Mice, 

TABLE D3 

Date Mixed Laboratoryb Laboratory' 

Study Referee 

Determined Concentration (mpJmL) 

Target Concentration 

(mg/mL)= 

18 March 1986 

26 November 1985 

5 August 1985 

18 April 1985 

Benzoyl Peroxide 

18 March 1986 

1 October 1985 

1 May 1985 

18 April 1985 

7,12-Dimethylbenz(a)anthracene 

14 April 1986 

28 October 1985 

17 May 1985 

N-Methyl-N'-nitro-N-nitrosoguanidine 

12 May 1986 

21 January 1986 

25 November 1985 

7 August 1985 

1 May 1985 

18 April 1985 

12-0-Tetradecanoylphorbol-13-acetate 

100 

100 

100 

100 

0.025 

0.025 

0.025 

0.025 

1 .o 

1.0 

1 .o 

0.01 

0.05 

0.01 

0.05 

0.05 

0.05 

97.20 

94.50 

98.67 

96.04 

106.3 f 0.6 

99.9 f 0.1 

80.9 f 1.9 

82.7 f 0.8 

0.02589 

0.02609 

0.02451 

0.024 

1.03 

0.96 

1 .00 

0.00904 

0.0468 

0.00998 

0.05326 

0.0519 

0.0537 

0.0242 f 0.1 

0.0249 f 0.0 

0.0239 f 0.0 

0.0208 f 0.0 

1.22 f 0.03 

0.98 f 0.02 

1.05 f 0.01 

0.0098 f 0.0 

0.0467 f 0.0002 

0.0097 f O.Ob 

0.0473 f 0.0004 

0.0387 f O.OOO3 

0.0375d 

a Dosing volume = 0.1 mL for 7,12-dimethylbenz(a)anthracene, N-methyl-N'-nitro-N-nitrosoguanidine, and 

single analysis 

Results of triplicate analyses 

Results of duplicate analyses 

0.1 mL) 

12-0-tetradecanoylphorbol-13-acetate. Dosing volume for benzoyl peroxide was 0.2 mL (two consecutive administrations of 
187 

AND CONTAMINANT LEVELS 

INGREDIENTS, NUTRIENT COMPOSITION, 

APPENDIX E 

IN NIH-07 RAT AND MOUSE RATION 

TABLE El Ingredients of NIH-07 Rat and Mouse Ration 

TABLE E2 Vitamins and Minerals in NIH-07 Rat and Mouse Ration 

TABLE3 Nutrient Composition of NIH-07 Rat and Mouse Ration 

TABLE E4 Contaminant Levels in NIH-07 Rat and Mouse Ration 

............................... 

190 

189 

188 

188 

...................... 

....................... 

........................ 
188 Skin Paint Studies, NTP TR 441 

Ingredients of NIH-07 Rat and Mouse Rationa 

TABLEl 

Ingredientsb Percent by Weight 

Premixes (vitamin and mineral) 

Salt 

Ground limestone 

Dicalcium phosphate 

Dry molasses 

Dried brewer’s yeast 

Soy oil 

Corn gluten meal (60% protein) 

Alfalfa meal (dehydrated, 17% protein) 

Dried skim milk 

Wheat middlings 

Fish meal (60% protein) 

Soybean meal (49% protein) 

Ground hard winter wheat 

Ground #2 yellow shelled corn 

10.00 

10.00 

12.00 

23.00 

24.50 

0.25 

0.50 

0.50 

1.25 

1.50 

2.00 

2.50 

3.00 

4.00 

5.00 

a NCI, 1976; NIH, 1978 

Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed. 

Vitamins and Minerals in NIH-07 Rat and Mouse Rationa 

TABLE E2 

Amount Source 

Vitamins 

A 

K3 

D3 

Thiamine 

Riboflavin 

d-Pantothenic acid 

Niacin 

Folic acid 

Choline 

d-a-Tocopheryl acetate 

B12 

Pyndoxine 

Biotin 

Cobalt 

Iodine 

Copper 

Zinc 

Manganese 

Iron 

Minerals 

18.0 g 

30.0 g 

2.2 g 

560.0 g 

20,000 IU 

2.8 g 

4,600,000 IU 

5,500,000 IU 

10.0 g 

3.4 g 

4,000 fig 

140.0 mg 

1.7 g 

120.0 g 

0.4 g 

1.4 g 

4.0 g 

16.0 g 

60.0 g 

Menadione 

D-activated animal sterol 

Stabilized vitamin A palmitate or acetate 

Choline chloride 

d-Calcium pantothenate 

Thiamine mononitrate 

d-Biotin 

Pyndoxine hydrochloride 

Cobalt carbonate 

Calcium iodate 

Copper sulfate 

Zinc oxide 

Manganous oxide 

Iron sulfate 

a Per ton (2,000 Ib) of finished product 
Feed Analyses 189 

Nutrient Composition of NIH-07 Rat and Mouse Ration 

TABLE E3 

Nutrient Deviation 

Mean 2 Standard 

Range Number of Samples 

Ash (% by weight) 

Crude fiber (% by weight) 

Crude fat (% by weight) 

Protein (% by weight) 

Amino Acids (% of total diet) 

Valine 

Qrosine 

Tryptophan 

Threonine 

Phenylalanine 

Methionine 

Lysine 

Leucine 

Isoleucine 

Histidine 

Glycine 

Cystine 

Arginine 

Essential Fatty Acids (% of total diet) 

Linolenic 

Lyoleic 

Vitamins 

Choline (ppm) 

Vitamin B,, (ppb) 

Biotin (ppm) 

Folic acid (ppm) 

Pyridoxine (ppm) 

Pantothenic acid (ppm) 

Niacin (ppm) 

Riboflavin (ppm) 

Thiamine (ppm) 

a-Tocopherol (ppm) 

Vitamin D (IU/kg) 

Vitamin A (IU/kg) 

Minerals 

Sulfur (%) 

Magnesium (%) 

Sodium (W) 

Chloride (%) 

Potassium (%) 

Phosphorus (%) 

Calcium (%) 

Zinc (ppm) 

Manganese (ppm) 

Cobalt (ppm) 

Chromium (ppm) 

Iodine (ppm) 

Iron (PP@ 

Copper (PP@ 

6.53 f 0.26 

3.48 f 0.58 

5.57 f 0.50 

21.97 f 0.43 

1.089 f 0.057 

0.677 f 0.105 

0.223 f 0.160 

0.896 f 0.055 

0.994 f 0.125 

0.437 f 0.115 

1.273 -+ 0.051 

1.972 f 0.052 

0.917 f 0.034 

0.580 f 0.024 

1.160 f 0.050 

0.306 f 0.075 

1.287 f 0.084 

0.277 f 0.036 

2.389 -+ 0.233 

19.80 f 1.52 

36.92 f 9.32 

4,450 f 1,382 

9?707 f 2,523 

30.30 f 3.60 

100.95 f 25.92 

7.92 f 0.93 

3,068 -+ 314 

40.14 f 20.04 

0.267 f 0.049 

2.51 f 0.64 

9.25 f 2.62 

11.50 k 2.40 

58.14 2 9.91 

92.24 f 5.35 

356.2 f 90.0 

0.274 0.063 

0.168 k 0.008 

0.315 f 0.344 

0.526 f 0.092 

0.887 k 0.067 

0.93 f 0.04 

1.16 f 0.10 

0.797 k 0.23 

1.71 f 0.45 

3.70 f 1.14 

6.13 - 6.97 

2.70 - 5.40 

4.70 - 6.40 

21.10 - 22.50 

0.962 - 1.170 

0.564 - 0.794 

0.107 - 0.671 

0.824 - 0.985 

0.665 - 1.110 

0.306 - 0.699 

1.200 - 1.370 

1.850 - 2.040 

0.867 - 0.965 

0.531 - 0.608 

1.060 - 1.220 

0.181 - 0.400 

1.100 - 1.390 

0.210 - 0.320 

1.830 - 2.570 

2,400 - 3,430 

10.6 - 65.0 

0.17 - 0.35 

1.80 - 3.70 

5.60 - 14.0 

23.0 - 34.6 

65.0 - 150.0 

6.10 - 9.00 

17.0 - 23.0 

22.5 - 48.9 

3,000 - 6,300 

6,200 - 15,000 

8.090 - 15.39 

46.10 - 81.60 

81.70 - 99.40 

255.0 - 523.0 

0.208 - 0.420 

0.151 - 0.180 

0.258 - 0.370 

0.380 - 0.635 

0.772 - 0.971 

0.87 - 0.99 

0.98 - 1.49 

0.490 - 1.150 

0.85 - 2.09 

1.52 - 5.83 

15 

15 

15 

15 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

9 

9 

9 

10 

10 

10 

10 

10 

9 

10 

15 

9 

4 

15 

10 

10 

10 

10 

10 

10 

10 

10 

8 

8 

15 

15 

6 

9 
190 Skin Paint Studies, NTP TR 441 

Contaminant Levels in NIH-07 Rat and Mouse Ration 

TABLE FA 

Deviationa 

Mean 2 Standard 

Range Number of Samples 

Cadmium (ppm) 

Arsenic (ppm) 

Contaminants 

(PPm) 

Nitrite nitrogen (ppm) 

Nitrate nitrogen (ppm 

Aflatoxins (ppb) 

Selenium (ppm) 

Mercury (PPm) 

b" 

BHT (PPm)" 

BHA (PPmY 

N-Nitrosopyrrolidine (ppb) 

N-Nitrosodimethylamine (ppb) 

Total nitrosoamines (ppb)f 

Salmonella (MPN/g) 

E. coli (MPN/g) 

Coliform (MPN/g)e 

Aerobic plate count (CFU/g)d 

Pesticides (ppm) 

a-BHCg 

6-BHC 

y -BHC 

B-BHC 

Endosulfan sulfate 

Endosulfan I1 

Endosulfan I 

Malathionh 

Ethyl parathion 

Methyl parathion 

Diazinon 

Trithion 

Ethion 

Ronnel 

Estimated PCBs 

Toxaphene 

Chlordane 

Telodrin 

Endrin 

Dieldrin 

Methoxychlor 

Mirex 

HCB 

DDT 

DDD 

DDE 

Heptachlor epoxide 

Aldrin 

Heptachlor 

0.76 f 0.14 

0.58 f 0.28 

co.10 

1.80 f 1.08 

2.47 f 0.99 

0.13 f 0.10 

14.92 f 4.41 

~5.0 

0.35 & 0.09 

CO.05 

c3.00 

4.40 rt: 2.41 

44,411 f 43,967 

1.16 f 0.62 

6.04 f 3.56 

7.20 & 3.84 

Negative 

<0.02 

co.02 

co.1 

<0.05 

eo.02 

co.01 

<0.2 

co.1 

<0.05 

co.01 

co.01 

co.01 

CO.05 

co.01 

co.01 

co.01 

co.01 

co.01 

co.01 

co.01 

eo.01 

co.01 

co.01 

co.02 

co.01 

<0.03 

co.01 

<0.01 

0.33 rt 0.81 

0.47 - 0.94 

0.14 - 1.32 

0.17 - 0.48 

<LOO - 4.00 

<2.00 - 5.00 

<0.10 - 0.50 

2.80 - 22.0 

3.00 - 9.00 

770 - 130,000 

1.00 - 3.40 

2.80 - 15.00 

3.80 - 16.00 

cO.05 - 3.20 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

1.5 

15 

15 

15 

15 

15 

15 

15 

15 

15 

15 

1.5 
Feed Analyses ,. 191 

TABLE E4 

Contaminant Levels in NIH-07 Rat and Mouse Ration (continued) 

a For values less than the limit of detection, the detection limit is given as the mean. 

CFU = colony forming unit 

Sources of contamination: soy oil and fish meal 

Sources of contamination: alfalfa, grains, and fish meal 

One lot milled on 7 May 1985 contained more than 0.7 ppm. 

g BHC is hexachlorocyclohexane or benzene hexachloride 

All values were corrected for percent recovery. 

e MPN = most probable number 
192 Skin Paint Studies, NTP TR 441 
193 

SENTINEL ANIMAL PROGRAM 

APPENDIX F 

and SENCAR Mice in the Comparative Initiatioflromotion Skin Paint Studies . . . . . 196 

TABLE F1 Murine Virus Antibody Determinations for B6C3Fl Mice, Swiss (CD-1) Mice, 

METHODS......,........... ................................... . ... .... ........ 194 
194 Skin Paint Studies, NTP TR 441 

SENTINEL ANIMAL PROGRAM 

source and weanling groups as the animals used for the studies of chemical compounds. 

are subject to identical environmental conditions. The sentinel animals come from the same production 

serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals 

evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via 

Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic 

optimally clean facilities to eliminate potential pathogens that may affect study results. The Sentinel 

Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in 

METHODS 

blood was collected during the studies are also listed. 

serology methods and viral agents for which testing was performed are tabulated below; the times at which 

Microbiological ASsociates, Inc. (Bethesda, MD), for determination of antibody titers. The laboratory 

collected, allowed to clot and the serum separated. The samples were processed appropriately and sent to 

virus was performed on sentinel mice in study design A of all three strains. Blood from each animal was 

collected from several moribund animals throughout the studies. A special screening for mouse hepatitis 

6 months, and from Acetone/Acetone control animals at study termination. Serum samples were also 

Serum samples were collected from randomly selected sentinel mice during the quarantine period and at 

Complement Fixation 

Method and Test 

B6C3Fl MICE 

LCM (lymphocytic choriomeningitis virus) 

ELISA 

MHV (mouse hepatitis virus) 

Mouse adenoma virus 

GDVII (mouse encephalomyelitis virus) 

Ectromelia virus 

Sendai 

Reovirus 3 

PVM (pneumonia virus of mice) 

Mycoplasma pulmonis 

Mycoplasma arthritidis 

Hemagglutination Inhibition 

Polyoma virus 

MVM (minute virus of mice) 

K (papovavirus) 

Immunofluorescence Assay 

EDIM (epizootic diarrhea of infant mice) 

Time of Analysis 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

study termination 

Quarantine, special screening, 6 months, 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 
Sentinel Animal Program 195 

Complement Fixation 

Method and Test 

SWISS (CD-l@) MICE 

ELISA 

LCM 

MHV 

Mouse adenoma virus 

GDVII 

Ectromelia virus 

Sendai 

Reovirus 3 

PVM 

M. pulmonis 

M. arthritidis 

Hemagglutination Inhibition 

Polyoma virus 

MVM 

K 

Immunofluorescence Assay 

Sendai 

Reovirus 3 

EDIM 

Complement Fixation 

Method and Test 

SENCAR MICE 

ELISA 

LCM 

MHV 

Mouse adenoma virus 

GDVII 

Ectromelia virus 

Sendai 

Reovirus 3 

PVM 

M. pulmonis 

M. arthritidis 

Hemagglutination Inhibition 

Polyoma virus 

MVM 

K 

Immunofluorescence Assay 

MHV 

MVM 

EDIM 

Quarantine, 6 months, study termination 

Time of Analvsis 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

study termination 

Quarantine, special screening, 6 months, 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Study termination 

Study termination 

Quarantine, 6 months, study termination 

Time of Analvsis 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

study termination 

Quarantine, special screening, 6 months, 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Quarantine, 6 months, study termination 

Special screening 

6 Months 

Quarantine, 6 months, study termination 
/ 

1’ 196 \/ Skin Paint Studies, NTP TR 441 

Results of serology tests are presented in Table F1. 

in the Comparative Initiatioflromotion Skin Paint Studies 

Murine Virus Antibody Determinations for B6C3F, Mice, Swiss (CD-la) Mice, and SENCAR Mice 

TABLE F1 

interval 

in Sentinel Animals 

Incidence of Antibody 

Reaction for 

Positive Serologic 

Study termination 

6 Months 

Special screening 

Quarantine 

B6C3F, Mice 

Swiss (CD-l@) Mice 

6 Months 

Special screening 

Quarantine 

Study termination 

7 Monthsa 

SENCAR Mice 

Study termination 

6 Months 

Special screening 

Quarantine 

0140 

OD 

on9 

on0 

0/10 

0/40 

1/20 

3/19 

ID 

213 

1/19 

in9 

2/30 

1/30 

1/26 

119 

0150 

None positive 

None positive 

None positive 

None positive 

Sendai 

Reovirus 3 

M. 

Mouse adenoma virus 

Sendai 

GDVII 

None positive 

None positive 

Reovirus 3 

M. arthritidi.sb 

EDIM 

MHV 

None positive 

a Evaluation of 3 moribund animals 

Accordingly, M. arthritidis positive titers were considered to be false positives. 

positive and there were no clinical signs or histopathologic changes of M. arthritidis infection in mice with positive titers. 

titers may be due to cross reaction with antibodies of nonpathogenic Mycoplasma or other agents. Only sporadic samples were 

Further evaluation of samples positive for M. arthritidis by immunoblot and Western blot procedures indicated that the positive 
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