Development of a transgenic mouse model for carcinogenesis ...

49, 241–254 (1999) Copyright © 1999 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Development of a Transgenic Mouse Model for Carcinogenesis Bioassays: Evaluation of Chemically Induced Skin Tumors in Tg.AC Mice Judson W. Spalding,* ,1 John E. French,* Raymond R. Tice,† Marianna Furedi-Machacek,†, Joseph K. Haseman,‡ and Raymond W. Tennant* *Laboratory of Environmental Carcinogenesis and Mutagenesis, and ‡Laboratory of Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and †Integrated Laboratory Systems, Research Triangle Park, North Carolina 27709 Received September 8, 1998; accepted January 27, 1999

Transgenic rodent models have emerged as potentially useful tools in the assessment of drug and chemical safety. The transgenic Tg.AC mouse carries an inducible v-Ha-ras oncogene that imparts the characteristic of genetically initiated skin to these animals. The induction of epidermal papillomas in the area of topically applied chemical agents, for duration of not more than 26 weeks, acts as a reporter phenotype that defines the activity of the test article. We describe here the activity of six chemicals that have been previously characterized for activity in the standard 2-year bioassay conducted by the National Toxicology Program (NTP). Homozygous female Tg.AC mice were treated with benzene (BZ), benzethonium chloride (BZTC), o-benzyl-p-chlorophenol (BCP), 2-chloroethanol (2-CE), lauric acid diethanolamine (LADA) and triethanolamine (TEA). BZ and LADA induced skin papillomas in a dose-dependent manner, while BCP induced papillomas only at the highest dose. BZTC, 2-CE, and TEA exhibited no activity. The correspondence of chemical activity in Tg.AC mice with that in the 2-year bioassay was high. A comparison of responsiveness to BZ and LADA was made between hemizygous and homozygous female Tg.AC mice. Both genotypes appear to be equally sensitive to maximum doses of active compounds. The results reported here indicate that the Tg.AC transgenic mouse model can discriminate between carcinogens and noncarcinogens and that both mutagenic and nonmutagenic chemicals can be detected. These studies provide support for the adjunctive use of the Tg.AC transgenic mouse skin tumor model in drug and chemical safety assessment and for the prediction of the carcinogenic potential of chemicals. Key Words: Tg.AC transgenic mice; chemical carcinogens; carcinogen bioassay; skin cancer.

For the better part of 4 decades, assessment of the carcinogenic potential of chemicals has largely relied upon the inter1

To mental Health 27709.

whom correspondence should be addressed at Laboratory of EnvironCarcinogenesis and Mutagenesis, National Institute of Environmental Sciences, PO Box 12233, MD F1-05, Research Triangle Park, NC Fax: (919) 541-1460. E-mail: [email protected].

pretation of chronic bioassays conducted in rodents. Despite steady improvement and standardization of study design and conduct, interpretation and extrapolation of these experimental results to human carcinogenic risk is often difficult. The current scheme, used by the National Toxicology Program (NTP), initially described by Huff et al., (1991) and more recently reviewed by Tennant (1993), compartmentalizes data according to species and sex, then stratifies experimental results as affording either clear, some, equivocal, or no evidence of carcinogenicity for each of the sex-species groups. The distinction between levels of evidence is determined primarily by the magnitude of the carcinogenic effect(s) observed. However, other factors such as background spontaneous tumor rates, the classification of the tumor in question (benign or malignant), and the presence or absence of a dose-response relationship are also considered. Although the currently performed chronic 2-year bioassays have been recognized as the most logical preclinical approach to chemical carcinogen identification, they are among the most time- and resource-intensive experiments that can be conducted, which severely limits the number of chemicals that can be evaluated. Clearly, there exists a need for improvement in the ways chemical carcinogens are identified. Short-term testing of chemicals in bacteria and cultured mammalian or nonmammalian cell models, while offering early promise as a means of reducing reliance on the bioassay, has remained in an adjunctive role in carcinogen identification. While these in vivo and in vitro models for assessing mutagenic potential have provided a wealth of information regarding mechanistic issues, their lack of biological complexity limits the predictive validity of results. Transgenic rodent models have recently emerged as potentially useful tools for assessing chemical carcinogenesis. Transgenic animals possess genetic alterations (such as the presence of specific oncogenes or absence of tumor suppressor genes) that are critical to the multi-stage process of tumorigen-

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esis, but, by themselves, are insufficient to induce cancer. The Laboratory of Environmental Carcinogenesis and Mutagenesis of the National Institute of Environmental Health Sciences has been conducting a systematic assessment and validation of transgenic mouse models for utility in the rapid identification of carcinogens. Two transgenic lines, Tg.AC and p53 (1/–)deficient mice, offer particularly attractive characteristics for incorporation into a battery of experimental methods that could be routinely employed for the assessment of carcinogenic potential (Spalding et al., 1993; Tennant et al., 1995, 1996). Tg.AC mice, initially described by Leder et al., (1990), carry a v-Ha-ras oncogene fused to the promoter of the z-globin gene. The combined effect of point mutations at codons 12 and 59 on the v-Ha-ras gene and the integration site of the transgene on chromosome 11 confers on these mice the characteristics of genetically initiated skin. Thus, the skin of the Tg.AC mouse is a target for tumorigenesis in the context of the well-known 2-stage mouse-skin tumorigenesis model (Boutwell, 1964, 1974; Hennings et al., 1993; Hennings and Yuspa, 1985). An important feature of the Tg.AC mouse model is that the transgene is not constitutively expressed in skin, which in untreated animals appears normal throughout their lifespan when compared to the skin of the wild type FVB/N parent strain (Hansen et al., 1996; Hansen and Tennant, 1994a). Further, the spontaneous incidence of tumors on the skin of untreated Tg.AC mice is very low (Spalding et al., 1993). The purpose of this communication is to present findings from assessments of skin-tumor responses of Tg.AC mice to dermally applied negative and positive control agents, as well as to selected chemicals of previously determined carcinogenic potential in rodents that were evaluated in the National Toxicology Program (NTP) 2-year bioassay. MATERIALS AND METHODS Experimental animals. Four- or 5-week-old female homozygous or hemizygous Tg.AC mice were received from an NTP-maintained colony at Taconic Farms Inc., Germantown, NY. Female mice were preferentially used for these studies, since they could be housed 4 –5/cage which was more cost effective than using males which would have had to be singly housed. The ages of the mice at initiation of treatment ranged from 7 to 18 weeks. Mice were maintained in groups of 4 or 5, in polycarbonate shoebox-style cages containing hardwood bedding (Beta Chips, Northeastern Products Corp., Warrensbury, NY). Husbandry practices were carried out in compliance with NIH Guidelines for Humane Care and Use of Laboratory Animals. Room temperature was 72 6 2°F; humidity ranged from 40 – 60%; and fluorescent lighting was on a 12-h light-dark cycle. Purina Pico Chow No. 5058 (Ralston Purina, St. Louis, MO) and tap water were available ad libitum. Animals were uniquely identified with tattoo numbers on their tails (Animal Identification and Marking System, Piscataway, NJ). Prior to beginning chemical exposure, 15–20 animals were randomly allocated to each treatment group, the mice remaining with their originally assigned cage mates. Chemicals. Ethanol (ETOH) was obtained from Pharmco Products, Inc., Bayonne, NJ; acetone (ACOH) from Mallinckrodt Speciality Chemicals Co., Paris KY; 12–0-tetradecanoylphorbol-13-acetate (TPA) and benzene (BZ) from Sigma Chemical Co., St. Louis, MO; benzethonium chloride (BZTC) from Midwest Research Institute, Kansas City, MO; 2-chloroethanol (2-CE),

from Aldrich Chemical Company, Milwaukee, WI; o-benzyl-p-chlorophenol (BCP), from Pflatz and Bauer, Waterbury, CT; and lauric acid diethanolamine (LADA), and triethanolamine (TEA) from the National Toxicology Program, NIEHS. Experiments conducted with chemicals of known bioassay results. Four chemicals (benzene, benzethonium chloride, 2-chloroethanol, and o-benzyl-pchlorophenol) of known history in the NTP bioassay program were selected for assessing the responsiveness of Tg.AC mice to topical administration. Two other chemicals, triethanolamine and lauric acid diethanolamine were on test in the 2-year bioassay and the studies in Tg.AC mice were completed before the 2-year studies were finished. Except for benzene, these chemicals were in dermal studies and the NTP bioassay results for all 6 agents are summarized in Table 1. Application of test chemicals. One to 3 days prior to the initiation of treatment on the application site, an area approximately 5–7 cm 2, (dependent on the age and weight of the mouse) from the dorsal interscapular region to about 1 cm from the base of the tail, was closely shaved with an electric shaver (Wahl series 8900, 1 inch No. 30 blade or an Oster Finisher Trimmer 76059 – 030, w/0000 blade). During the experiment, mice were shaved, as necessary, until the presence of tumors made it inadvisable. Care was taken during shaving to avoid abrasions or cuts but occasionally superficial nicks in the skin occurred. Tumors attributable to superficial lesions caused by shaving were rarely observed. Dose selection, preparation, and application. Test agents were solubilized in acetone or 70 –95% ethanol and applied topically in a total volume of 200 ml with an automatic pipette (Lab Systems series 4501). That volume, when applied in an anterior to posterior manner along the dorsal midline, spreads evenly over the shaved area. The frequency of administration of the appropriate, concurrent negative control solvent matched that of the test agent. The concurrent positive control agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), was administered at a dose of 1.25 mg or 1.5 mg in 200 ml of ACOH 2–3 times weekly, e.g., either Monday and Thursday or Monday, Wednesday, and Friday for 20 weeks. This relatively low dose of TPA was selected because, in our lab, it is consistently tumorigenic in female homozygous Tg.AC mice, but usually does not cause a maximal response during the 20-week treatment period. The chemical doses selected for these studies were based on the dose range used for female mice in the 2-year NTP dermal-exposure studies. The highest of the 3 doses used was equal to or exceeded the NTP maximum tolerated dose (MTD). In the case of BZTC, 2-CE, LADA, and TEA, higher than MTD doses were selected because those doses had not reduced survival or caused toxicity in the NTP 13-week subchronic dose-finding study. Benzene was applied in doses of 200 (7.0 gm/kg), 400, or 800 ml, twice per week. To achieve this weekly dosage regimen, benzene was applied 2 days per week. This dose regimen was based on early reports (Boutwell, 1957 and Van Duuren, 1965) in which benzene was often the solvent of choice used for topical chemical administration in the 2-stage mouse skin tumorigenesis model. Volumes of 100 –200 ml were applied 2–3 times per week for up to 70 weeks and no tumors or severe irritation were reported for the benzene solvent control groups. Benzethonium chloride was dissolved in 95% ethanol and 10, 30, or 60 mg (0.4, 1.2, and 2.4 mg/kg) were applied 5 days/week for 20 weeks. These doses were in the range of the 0.15–1.5 mg/kg doses applied dermally in the NTP study (NTP, 1995a). 2-Chloroethanol was dissolved in 70% ethanol and applied in doses of 5.0, 10.0, or 20.0 mg/day, 5 days/week for 20 weeks. These dose levels encompassed the range of doses applied to the backs of male and female CD-1 mice (7.5 or 15 mg) used in the NTP bioassay (NTP, 1985). O-Benzyl-p-chlorophenol was dissolved in acetone and doses of 0.1, 1.0, or 3.0 mg were applied 3 days/week for 20 weeks. This dose range and frequency was the same as that used for the 1-year skin paint study in CD-1 mice performed by the NTP (NTP, 1995b). Triethanolamine was solubilized in acetone and administered at doses of 3.0, 10.0, or 30.0 mg, (120, 400 and 1200 mg/kg) 5 days/week for 20 weeks. The

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TABLE 1 Summary of the Results of NTP Assessments of Toxicologic and Carcinogenic Potentials of Benzene, Benzethonium Chloride, 2-Chloroethanol, o-Benzyl-p-chlorophenol and Triethanolamine

Compound (CAS No.) Benzene (71-43-2)

Route

Species and dose level

5344/N rats M 50, 100, 200 mg/kg F 25, 50, 100 mg/kg B 6C 3F 1 mice M&F 25, 50, 100 mg/kg Benzethonium chloride Dermal application F344/N rats and B 6C 3F 1 (121-54-0) in acetone 5 days per mice 0.15, 0.5, 1.5 mg/kg week 2-Chloroethanol Dermal application F344/N rats (107-07-3) in 70% ethanol 5 M&F 50, 100 mg/kg days per week Swiss CD-1 mice M&F 7.5, 15 mg/dose o-Benzyl-p-chlorophenol Gavage in corn oil, 5 F344/N rats (120-32-1) doses per week M 30, 60, 120 mg/kg F 60, 120, 240 mg/kg B 6C 3F 1 mice M&F 120, 240, 480 mg/kg Dermal application Swiss CD-1 mice in acetone 3 days per 0.1, 1.0, 3.0 mg/dose week for 52 weeks Triethanolamine Dermal application in F344/N rats (102-71-6) acetone, 5 days per M 32, 63, 125 mg/kg week F 63, 125, 250 mg/kg

Lauric acid diethanolamine (120-40-1)

Gavage in corn oil, 5 times per week

Dermal application in 95% ethanol, 5 days per week

B 6C 3F 1 mice M 200, 630, 2000 mg/kg F 100, 300, 1000 mg/kg F344/N rats M&F 50, 100 mg/kg B 6C 3F 1 mice M&F 100, 200 mg/kg

Evidence of carcinogenicity a

Evidence of mutagenicity b

Reference

CE in both sexes and Increased MN in mice both species Negative in SAL

NTP TR No. 289, 1986

NE in either sex of either species

Negative in SAL, SCE and CHO HGPRT

NTP TR No. 438, 1995a

NE in either sex of either species

Positive in SAL TA 100 and TA 1535; negative in DROS SLRL Negative in SAL, SCE and CHO HGPRT

NTP TR No. 275, 1985

NE in male rats; EE in female rats; SE in male mice; NE in female mice; Skin tumor promoter

NTP TR No. 424, 1994

NTP TR No. 444, 1995b

EE in male rats: NE in female rats; EE in male mice; c SE in female mice c

Negative in SAL, SCE NTP TR No. 449, and CA in CHO cells, (in press) and MN in mice

NE in male rats NE in female rats NE in male mice SE in female mice

Negative in SAL, CA NTP TR No. 480, in CHO cells and MN (in press) in mice; positive in SCE in CHO cells

a

CE, clear evidence; SE, some evidence; NE, no evidence; EE, equivocal evidence. MN, micronucleated erythrocyte; SAL, Salmonella typhimurium; DROS SLRL, Drosophila melanogaster sex linked recessive lethal; SCE, sister chromatid exchange in Chinese hamster ovary cells; CHO HGPRT, Chinese hamster ovary hypoxanthine guanine phosporibosyl transposegene; CA, chromosomal aberrations in Chinese hamster ovary cells. c Results possibly compromised due to Helicobacter hepaticus infection. b

high dose of 30.0 mg was 20% greater than the highest dose (1000 mg/kg in female mice) used in the NTP dermal study (Table 4) (NTP, 1999b). Lauric acid diethanolamine was dissolved in 95% ethanol and applied in doses of 5.0, 10.0 or 20.0 mg, (200, 400 and 800 mg/kg) 5 days/week for 20 weeks. The 2 higher doses exceeded the highest dose used in the NTP bioassay. The peer-reviewed report for LADA is currently in press (NTP, 1999a). Animal observation. Mice were observed twice a day for mortality and morbidity. Body weights were recorded weekly, and the results of detailed clinical observations, including an examination of the site of application, were recorded 2–5 times weekly. Recorded observations included all evidence of systemic or local toxicity, the number of skin tumors (recorded weekly), and the presence of odontomas or malocclusions. Skin tumor responses were evaluated with knowledge of what animals comprised the different treatment groups. Tg.AC mice can develop spontaneous jaw tumors (odontomas) that may interfere with their ability to eat (Wright et al., 1995). These tumors begin to

appear at around 6 months-of-age, and any animals with odontomas were removed from the study. We have found no evidence that treatment with either negative or positive control agents or test agents influences the incidence of odontomas (unpublished observations). As the number of skin tumors increases in response to an active chemical or TPA (the positive control agent), accuracy in counting papillomas becomes problematic, since individual tumors that are close together tend to fuse as they increase in size. For this reason, an upper limit of 32 tumors per animal was arbitrarily observed during these experiments. While animals that had reached this limit were continued in their dosing regimen, no attempt was made to accurately count lesions. In fact, although we continued dosing all animals throughout the 20-week studies, it became unnecessary to continue exposures or observations once an unequivocally positive response to either the test agent or TPA had been established. Criteria for grading tumor responses. In order to be counted as a positive response, skin tumors had to reach 1 mm in size and persist for at least 3 weeks. In practice, since individual tumors were not mapped, this requirement applies only to the first few tumors that appear, because as the papilloma

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TABLE 2 Skin Tumor Incidence in Homozygous Female Tg z AC Mice Topically Dosed for 20 Weeks with Benzene

Treatment Acetone Benzene b 400 ml/wk 800 ml/wk 1600 ml/wk TPA c 2.5 mg/week

Incidence of animals with tumors (%)

Mean weeks to first tumor (6 SD)

Mean tumors per animals at risk (6 SD) a

Mean weeks to maximal tumor yield (6 SD)

Survival at 20 weeks (%)

3/5 (60%)

14.0 6 2.6

1.4 6 1.7

14.3 6 2.9

4/5 (80%)

7/10 (70%) 8/10 (80%) 10/10 (100%) d

12.6 6 6.1 9.1 6 1.4 8.4 6 1.8

7.0 6 10.3 10.6 6 8.5 e 12.6 6 10.3 e

18.4 6 2.3 16.5 6 2.9 14.3 6 3.7

8/10 (80%) 8/10 (80%) 8/10 (80%)

13.0 6 3.9

13.0 6 11.4 e

19.0 6 2.2

5/5 (100%)

5/5 (100%)

Note. Mice were 7 weeks old at start of treatment. a Animals were considered to be at risk after 10 weeks of dosing. b Benzene was applied 2 days per week; half the daily dose was administered during the morning and half during the afternoon. The highest dose was applied as 2 successive 200 ml applications of neat benzene in the morning and 2 in the afternoon. The mid-dose was applied in 200 ml doses of neat benzene during the morning and afternoon. The low dose was applied as a 1:1 solution of benzene in acetone with 200 ml/day, morning and afternoon of dosing days. c TPA (1.25 mg) in 200 ml acetone was applied twice per week. d p , 0.05 vs. acetone controls (Life Table Analysis). e p , 0.05 vs. acetone controls (Mann-Whitney U Test).

multiplicity increases on any one mouse, some papillomas may regress and others appear. Transient papillomas that appeared in the application area for only 1 or 2 weeks were deleted from the computations. Loss of papillomas due to sloughing or scratching was observed and more often occurred in animals with high (15–30) papilloma loads. Alternatively, we occasionally observed true regression over the course of a study, and this could occur even though treatment of the animal was discontinued. The latency time for the first tumor, and the tumor burden for each mouse in any dose group, were recorded weekly. Animals that did not survive the first 10 weeks of treatment were deleted from the study. In order to compare papilloma responses among different treatment groups, the maximum papilloma burden of each animal by the end of 20 weeks was the value used in the computation. However, some exceptions to the above stated guidelines were made as follows: ● If a papilloma appeared during weeks 18, 19, or 20, it was tabulated, even though it had not been present for 3 weeks. ● Animals that were removed from the study between 11 and 20 weeks were given the maximum tumor burden that occurred during their lifetime in the study. ● When tumor regression occurred, an average of the highest tumor burdens over 3 consecutive weeks was the value used in the computation. ● In studies with active chemicals and in the TPA positive control we typically saw a range of responses among the individual animals within any dose group. Low and high responders occur most often at chemical doses that appear on the linear part of a dose-response curve. However, at more optimal inducing doses, e.g., 1.25 mg TPA, 33/week, or 1.5 mg TPA, 23/week, individual animal papilloma burdens clustered in the range of 20 –32 or more (see Fig. 3).

Duration of studies. Dosing was terminated after 20 weeks, and the animals were often held for observation for an additional 6 to 10 weeks before study termination. Despite the fact that some animals were observed for additional periods, only data collected during the initial 20-week experimental period have been used for this communication. During the post-dosing and observation periods, interim sacrifices and necropsies were conducted at irregular intervals. In some studies, blood and tissue samples were taken at the end of the 20-week dosing period and/or termination of the study at 30 weeks. These samples were preserved for later analysis, the results of which will be published elsewhere.

None of the doses selected for these studies was observed to cause overt skin irritation or ulceration as determined by gross examination. Skin biopsies were not routinely taken from the treated mice, since they were held 6 to 10 weeks after the last treatment for observation, and it was not expected that histopathological examination of skin biopsies taken at terminal sacrifice would reveal useful information, especially among mice treated with inactive test agents. The epidermal papillomas induced by BCP, BZ, and LADA did not differ in gross appearance from those induced by TPA or wound repair. The histopathological characteristics of the TPA and wound-repair-induced papillomas have been reported previously (Cannon et al., 1997; Hansen and Tennant, 1994a,b). Statistical analyses. Two statistical procedures were used to evaluate the skin papilloma data. The first was a life table test (Cox, 1972; Tarone, 1975) based on the time of initial tumor onset for each animal. This procedure was used both to assess dose-response trends across groups and to compare each dosed group to the corresponding controls. To evaluate tumor multiplicity, maximum papilloma yield for each animal was calculated and dose-response trends evaluated by Jonckheere’s test (Jonckheere, 1954). Pairwise comparisons of each dosed group with controls was made by the Mann-Whitney U test (Siegel, 1956).

RESULTS

Responses in the Negative and Positive Control Groups Homozygous Tg.AC mice in the negative control groups treated with acetone or 70 –95% ethanol (Figs. 1 and 2) developed a very low frequency of papillomas. The incidence of papilloma induction in the negative control groups ranged from zero to 29%. Rarely did a vehicle-treated animal develop more than a single papilloma, and with the exception of the benzene study (Table 2), the average (mean) maximum papilloma multiplicity for the negative control groups was always less than 1.0. Among the 67 animals that comprised the different solvent control groups reported here, the incidence of papilloma-bearing mice was 10% (7/67). These 7 mice developed one tumor each, so that the average multiplicity for the negative controls

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TABLE 3 Skin Tumor Incidence in Homozygous Female Tg.AC Mice Topically Dosed for 20 Weeks with Benzethonium Chloride or 2-Chloroethanol

Treatment Ethanol b BZTC c 10.0 mg/day 30.0 mg/day 60.0 mg/day 2-CE 5.0 mg/day 10.0 mg/day 20.0 mg/day TPA d 1.25 mg


Mean weeks to first tumor ( 6 SD)

2/15 (13%)

13.0 6 8.5

4/14 (29%) 0/15 (0%) 6/15 (40%)

Mean tumors per animals at risk ( 6 SD) a

Mean weeks to maximal tumor yield ( 6 SD)


0.1 6 0.0

13.0 6 8.5

14/15 (93%)

11.5 6 5.1 – 12.0 6 2.9

0.3 6 0.0 0.0 6 0.0 0.7 6 1.0 d

11.5 6 5.1 – 15.8 6 3.4

12/14 (86%) 13/15 (87%) 12/15 (80%)

2/15 (13%) 0/15 (0%) 5/15 (33%)

9.0 6 2.8 – 11.6 6 6.3

0.1 6 0.0 0.0 6 0.0 0.4 6 0.4

9.0 6 2.8 – 13.4 6 7.0

14/15 (93%) 14/15 (93%) 13/15 (87%)

14/15 (93%) f

12.4 6 4.1

11.1 6 8.2 e

19.9 6 4.2

13/15 (87%)

Note. Mice were 10 weeks old at start of treatment. a Animals were considered to be at risk after 10 weeks of dosing. b 95% ethanol was applied in doses of 200 ml, 5 days per week. c Benzethonium chloride was dissolved in 95% ethanol and 2-chloroethanol was dissolved in 70% ethanol. Both were applied 5 days per week. d TPA (1.25 mg) in 200 ml acetone was applied twice per week. e p , 0.05 vs. ethanol controls (Mann-Whitney U Test). f p , 0.05 vs. ethanol controls (Life Table Test).

was 0.10. In several instances, the time to first tumor or to maximum tumor yield (13–14 weeks) was similar to that seen for TPA (the positive control)-induced tumors. Survival of the negative control groups after 20 weeks ranged from 80 –100% and did not differ significantly from that observed among the chemical treatment groups. The high incidence and multiplicity of tumors seen in the acetone control group of the benzene study (Table 2) were wound-induced and caused by fighting among cage mates. After the mice were randomized by weight, they were reassigned to different cage-mate groups just prior to initiating treatment. This practice was discontinued in all of the other studies reported here. Even under optimal conditions of multiple housing of female mice, we have observed a clustering or “cage effect” among mice treated with the negative control solvent or an inactive test agent. A dominant female in a particular cage will inflict bite wounds on 2–3 cage mates, who will develop one or more papillomas at the wound sites. Since this cage dominance is established early, the wound repairinduced papillomas usually appear early in the study. The standard positive-control dose of TPA used in treating homozygous female Tg.AC mice was 1.25 mg, 23/week, for 20 weeks. The incidence of animals with papillomas ranged from 93–100% (Tables 2– 4), and the average maximum multiplicity per group ranged from 11 to 19.5 papillomas per mouse. This particular dose regimen was selected to induce a reproducible tumor response, but not a maximum response, e.g., 32 or more papillomas per mouse, in every individual animal. The range of tumor burdens among individual TPA-

treated mice ranged from 1 to over 32 papillomas per animal. Effects of Application of Test Agents on Skin Tumor Incidence in Tg.AC Mice Benzene. Dermal administration of benzene induced a dose-related increase in skin tumors and a concomitant decrease in tumor latency (Table 2). The average multiplicity (mean maximal number of tumors per animal at risk [animals dosed for at least 10 weeks]) increased in a dose-related manner. The time required to develop a maximal tumor yield decreased in a dose-related manner. The sensitivity of the statistical analysis for benzene was limited by the small number of acetone controls (5 animals) and the relatively high papilloma incidence (60%) in this group (Table 2). Nevertheless, life table analysis revealed a significant (p , 0.01) carcinogenic effect in the 1600-ml/week benzene group relative to acetone controls, due primarily to the earlier onset of skin papilloma in dosed animals. Nine of the 10 animals developing skin papillomas in the 1600-ml/week benzene group developed their initial papillomas during weeks 6 –9, whereas the 3 control animals with papillomas developed these tumors during weeks 11–16. Tumor multiplicity was also significantly increased in both the 800- (p , 0.05) and 1600-ml/week (p , 0.01) benzene groups. Perhaps because of the small sample sizes, the increase in tumor multiplicity in the 400-ml/week benzene group was not statistically significant. However, since 4 of the 10 animals in the 400-ml/week benzene group had maximum papilloma yields (26, 23, 9, and 8 papillomas), well

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TABLE 4 Skin Tumor Incidence in Homozygous Female Tg.AC Mice Topically Dosed for 20 Weeks with o-Benzyl-p-chlorophenol or Triethanolamine

Treatment Acetone BCP b 0.1 mg 1.0 mg 3.0 mg TEA 3.0 mg 10.0 mg 30.0 mg TPA e 1.25 mg


Mean weeks to first tumor (6 SD)

Mean tumors/animals at risk (6 SD) a

Mean weeks to maximal tumor yield (6 SD)


4/14 (29%)

13.3 6 2.1

0.3 6 0.0

13.3 6 2.1

14/14 (100%)

5/15 (33%) 1/13 (8%) 16/19 (84%) c

8.6 6 6.1 16.0 10.9 6 3.6

0.7 6 1.2 0.1 2.3 6 1.9 d

16.0 6 1.9 16.0 13.5 6 3.6

13/15 (87%) 10/13 (77%) 13/19 (68%)

4/14 (29%) 5/13 (38%) 4/19 (21%)

11.8 6 7.3 13.4 6 4.9 8.8 6 2.6

0.4 6 0.5 0.8 6 2.2 0.5 6 1.7

13.8 6 6.7 14.8 6 5.4 13.5 6 4.4

11/14 (79%) 12/13 (92%) 15/19 (79%)

19/20 (95%) c

7.4 6 2.4

19.5 6 12.4 d

13.6 6 3.5

9/20 (45%)

Note. Mice were 18 weeks old at start of treatment. a Animals were considered to be at risk after 10 weeks of dosing. b o-Benzyl-p-chlorophenol was dissolved in acetone and applied 2 times per week. Triethanolamine was dissolved in acetone and was applied 5 times per week. c p , 0.01 vs. acetone controls (Life Table Test). d p , 0.01 vs. acetone controls (Mann-Whitney U -Test). e TPA (1.25 mg) in 200 ml acetone was applied twice per week.

in excess of the numbers found in the acetone control group (1, 2, or 4 papillomas in 3 of 5 mice), this response is also considered to be benzene-related. Benzethonium chloride (BZTC). Neither the 10- nor the 30-mg/day BZTC-treated groups showed evidence of carcinogenicity (Table 3). The 3-fold increase in tumor incidence in the 60-mg/day group (40% vs. 13%) was not statistically significant by life table analysis. However, tumor multiplicity was marginally increased (p 5 0.04) by a one-sided Mann-Whitney U test. This marginal effect reflected the fact that 3 of the 6 animals with papillomas in the 60-mg/day BZTC group had multiple papillomas. However, both of the animals with papillomas in the ethanol control group had only a single papilloma, one of which developed relatively late in the study (week 19). A “cage effect” cannot be ruled out, since 3 of the 6 mice were cage mates and the papillomas could have resulted from bite wounds inflicted by a dominant female. With the exception of the benzene study, the average papilloma multiplicity among negative control groups, or in groups treated with inactive chemicals, has always been less than 1.0. Because of the variability of the “cage effect” in skin papilloma response, the marginal effect in the high-dose BZTC group was considered a spurious finding. 2-Chloroethanol (2-CE). There was no carcinogenic effect in the mice treated with 2-chloroethanol. Daily dermal applications of up to 20 mg of 2-CE had no effect, either on body weight gain (data not shown) or survival of Tg.AC mice, in this experiment (Table 3). Examination of the application sites revealed no gross evidence of local toxicity. The incidence and

average papilloma multiplicity of the 5 and 10 mg/kg treatment groups was no different from that observed in the negative (ethanol) control group. The slight increase in skin papillomas that was observed in the 20-mg/kg 2-CE group was also not statistically significant (Table 3). o-Benzyl-p-chlorophenol (BCP). Dermal applications of 0.1, 1.0, and 3.0 mg of BCP caused a dose-related decrease in survival time, with 6/19 (32%) mice in the high-dose group dying before the end of the experiment (Table 4). Mean survival time of the high-dose mice was 18.6 6 2.5 weeks compared to 20 6 0 weeks for the acetone-treated control group. Three applications per week of 3 mg were also associated with a significant (p , 0.01) carcinogenic effect by life-table analysis. Tumor multiplicity was also significantly (p , 0.01) increased in the 3.0 mg BCP group, although much less than that observed for benzene (compare Tables 2 and 4). The mean latency time to maximum tumor yield was the same for the negative and positive control groups and for the highest BCP dose group. Triethanolamine (TEA). The average papilloma incidence among animals treated 5 days per week with 3.0 to 30 mg of triethanolamine was not significantly different from the incidence observed in animals treated with acetone, the negative control and solvent vehicle (Table 4). At the end of 20 weeks, one animal in each of the 2 higher dose groups had 6 or 5 papillomas, respectively. It is of interest to note that among the animals in the high-TEA-dose group, 3 of the 4 mice in 1 cage accounted for 90% (9/10) of the total papillomas tabulated for that dose group. This pattern of response is a cage effect and is recurrent among the different studies. The most likely expla-


nation is that papilloma-bearing mice were the subjects of wounding by the dominant female in the cage. Because of this recurring pattern, we now recommend and practice single housing for these studies. There was no significant difference in weight gain among vehicle-control or TEA-treated groups (data not shown). Survival was slightly reduced in the TEAtreated groups, but this was mostly due to removal of animals with odontomas from the study. A Comparison of Papilloma Induction Response between Female Hemizygous and Homozygous Tg.AC Mice The data discussed so far were derived from experiments performed in homozygous female Tg.AC mice. Our earliest work with the Tg.AC mouse model was performed in hemizygous Tg.AC transgenic mice (Spalding et al., 1993). When it became apparent that homozygous Tg.AC mice exhibited similar fertility, longevity and spectrum of spontaneous tumors, we began conducting our experiments in homozygous mice, because genotyping of individual mice was not required (Spalding et al., 1993). This earlier work indicated that topically applied TPA, benzoyl peroxide, or butanone peroxide readily induced skin papillomas in both hemizygous and homozygous Tg.AC mice. There was little difference in the magnitude of response, but in these early experiments, the chemical doses selected were those that could be expected to induce a maximum tumor incidence. The result of an experiment comparing TPA-induced papilloma incidence and multiplicity between hemizygous and homozygous Tg.AC mice suggested that a transgene-dose effect might explain the data. Three different doses of TPA were administered twice a week for only 10 weeks. Although there was no difference in tumor incidence and multiplicity between the hemizygous and homozygous mice at the lowest dose (no-effect level) or the highest dose (maximum-level effect), a transgene-dose effect was seen at the mid-dose (2.5 mg). The maximal papilloma incidence was reached earlier and was higher in the homozygous mice (data not shown). Since the NTP had recently initiated studies using hemizygous Tg.AC mice (Eastin et al., 1998), we decided to compare the response of hemizygous mice to several chemicals that we had previously tested in homozygous mice, using the same doses and protocol. The response among the individual mice of both genotypes to topical treatment with 200-ml benzene, 3 times per week for 20 weeks, is compared in Figure 1. Papillomas were induced in 100% of the mice in both genotypes. At the end of 20 weeks, the papilloma multiplicity reached or exceeded 32 papillomas/mouse in all but one animal among the homozygous mice. Fifty-seven percent (8/14) of the hemizygous animals had papilloma loads of 32 or more, and the multiplicity among the other mice ranged from 3 to 25 papillomas per animal. However, homozygous mice developed these high papilloma yields significantly (p , 0.01) sooner than did hemizygous mice (Fig. 1). For example, at week 10,

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the mean number of papillomas per animal was approximately 3 times greater in the homozygous group than in the hemizygous group (data not shown). Moreover, all 13 homozygous mice still alive at week 15 had achieved a maximum papilloma response (32) compared with only one of the 12 surviving hemizygous animals (data not shown). The responses of both genotypes to the acetone control were very similar; no papillomas were seen in the acetone-treated homozygous group and only one mouse in the heterozygous group developed a papilloma, which appeared at week 18 of the study (Fig. 1). The average maximal papilloma burden per mouse induced by TPA, the positive control, was higher in the hemizygous mice than in the homozygous animals, but this was due to a slightly different dosing protocol, which used a higher dose (1.5 mg, 23/week) of TPA. When the standard TPA-dosing regimen of 1.25 mg 23/week, was continued for 20 weeks, 14 of 15 homozygous mice developed papillomas; and the range of response was 2–30 tumors per mouse (Fig. 1). When the dose of TPA was changed to 1.5 mg, twice a week, a dose increase of 20% per week, 60% of the treated hemizygous mice had a papilloma burden that reached or exceeded 32 papillomas per mouse (Fig. 1). Papillomas were induced in 80% and 93% of the hemizygous and homozygous mice, respectively. The 20-week survival rate was greater (86–100%) among all groups of hemizygous mice than that observed in homozygous mice. The lowest survival (67%) was seen in the benzene-treated homozygous group and was due primarily to removal of animals with odontomas from the study. Doses of 5, 10, and 20 mg of lauric acid diethanolamine (LADA) in 200 ml of 95% ethanol were administered topically 5 times per week for 20 weeks to 14-week-old female hemizygous Tg.AC mice. This was the same dosing protocol used for a previous experiment in 10-week-old homozygous Tg.AC mice. A comparison of the results of these 2 experiments is shown in Figure 2. A dose response was clearly indicated in the hemizygous treated animals. A dose-related response was also seen among the treated homozygous mice, but there was no significant difference in the average papilloma multiplicity at the 10- or 20-mg doses. The papilloma response at the 10- and 20-mg doses of LADA in both genotypes was significantly (p , 0.01) different from the concurrent, solvent negativecontrol groups. The maximum papilloma response among homozygous mice at the 10-mg dose was 4 times greater (p , 0.01) than that seen in the hemizygous group. There was no difference in response between the 2 genotypes at the highest dose (Fig. 2). One mouse in each of the solvent negativecontrol groups developed a single papilloma. The mean papilloma multiplicity in hemizygous mice treated with a slightly higher dose (1.5 mg, 23/week) of TPA was 3 times greater than that induced in homozygous mice treated with TPA 1.25 mg, 23/week (Fig. 2). In addition, the mean latency period was reduced by 3 to 4 weeks in the hemizygous mice treated with the higher weekly dose of TPA (data not shown). The variability in the magnitude of response among ho-

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FIG. 1. A comparison of the distribution of benzene-induced papilloma incidence in individual female hemizygous or homozygous Tg.AC mice. Fourteen-week-old hemizygous and 11-week old homozygous mice were topically dosed with 200 ml of benzene, 33/week for 20 weeks. Hemizygous mice treated with 1.5 mg of TPA, 23/week, and homozygous mice treated 23/week with 1.25 mg of TPA served as the positive control groups. The data depicted for the benzene-treated groups represent the maximal papilloma burden achieved by each mouse at the end of the 10- and 20-week-exposure periods. The values depicted for the acetone- and TPA-treated groups represent the maximal papilloma burdens of each mouse achieved during the 20-week-exposure period.

mozygous Tg.AC to the minimal dosing regimen with TPA, 1.25 mg, twice a week for 20 weeks, prompted an investigation into the reproducibility of TP-induced responses in hemizygous mice. The results from 4 independent studies are compared in Figure 3. Each group served as the positive control for 4 independent chemical studies. Two groups (A and B) received 1.25 mg TPA, 3 times a week for 20 weeks; the other 2 groups (C and D) received 1.5 mg TPA, twice a week for 20 weeks. The total TPA dose per animal per week was 3.75 mg and 3.0 mg respectively. There was no significant difference in the average papilloma/mouse response among the 4 groups. Among the 49 mice in the combined groups, 47 (96%) had papillomas; 36 mice (73%) had 20 or more papillomas. It is clear that hemizygous Tg.AC mice respond to TPA in a reproducible manner. When the tumor response of hemizygous and homozygous Tg.AC mice exposed to similar chemical treatment is compared, there is some evidence of a transgene-dose effect at dose levels that do not induce a maximal response. However, in no

case did hemizygous mice fail to respond to a chemical that induced papillomas in homozygous mice. Furthermore, at the maximal doses of LADA (20 mg), benzene (200 ml) or optimal doses of TPA (3.0 to 3.75 mg/week), nearly all of the mice in the dose groups of both genotypes responded. These data indicate that if dose levels are properly chosen there should be no appreciable difference in the capability of the hemizygous and homozygous genotypes to identify an active chemical. DISCUSSION

The validation process for any new alternative testing model should include the evaluation of chemical activity for both known carcinogens and non-carcinogens. With the exception of benzene, the chemical selection for these studies was based on the fact, that the chemicals had been subjects of 2-year dermal studies in the NTP bioassay. Though the number of chemicals was limited, they represented agents that had been defined by the Salmonella mutagenicity assay as genotoxic or


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FIG. 2. A comparison of the distribution of lauric acid diethanolamine-induced papilloma incidence in individual female hemizygous and homozygous Tg.AC mice. Fourteen-week-old hemizygous and 10-week-old homozygous female Tg.AC mice were topically dosed with 5, 10, or 20 mg of lauric acid diethanolamine (LADA), 53/week for 20 weeks. The hemizygous TPA-positive control group was the same as that described in Figure 1. The homozygous positive-control group was treated with 1.25 mg of TPA, 23/week for 20 weeks. The negative control groups received 200 ml of 95% ethanol, the solvent vehicle, 53/week for 20 weeks. The data depicted represent the maximal papilloma burden achieved by each mouse by the end of the 20-week-exposure period.

non-genotoxic carcinogens and non-carcinogens. Benzene was chosen because it was a known human and rodent carcinogen. It was of additional interest because, in the early studies of mouse skin carcinogenesis, it was often the solvent vehicle of choice for topical chemical administration and had shown no activity by itself in those studies (Boutwell et al., 1957; Van Duuren et al., 1965) at dose/frequency regimens similar to those used in the studies reported here. The responses to negative and positive control agents are important components in assessing and monitoring the performance of any test-model. It is desirable that the negative control substance, usually the solvent vehicle, consistently elicits no response during the exposure period. It is equally important to select a positive control substance that induces a reproducible response to a consistent dose/frequency protocol. TPA has been the positive control article in our studies, because the range of responses to different dose/frequency regimens has been well-documented (Spalding et al., 1993; Hansen and Tennant, 1994a).

The spontaneous papilloma incidence and multiplicity among Tg.AC mice at the site of application in untreated or negative (vehicle) control groups is low. The tumor incidence among the female homozygous mice exposed to either acetone or 95% ethanol was 7/67 or 10.5%. The total number of papillomas for this group was 7 (one papilloma on each mouse) or an average multiplicity of 0.10 for the animals at risk. The tumor incidence (2/37, 5.0%) and average multiplicity (2/37, 0.05) among the hemizygous, negative-control groups was also very low. The only exception to the low incidence and multiplicity among solvent control animals occurred in the benzene experiment where the animals were reassigned with different cage mates just prior to the start of treatment. This event led to excessive fighting and biting among the female mice as dominance was reestablished and resulted in wound site-induced papillomas. In all subsequent studies, the original cage assignments, established when the mice were received from the vendor, were maintained for the duration of the experiment.

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FIG. 3. Distribution of TPA-induced papilloma burdens in individual hemizygous female Tg.AC mice. Four groups of 14-week-old, hemizygous female mice served as TPA-treated positive controls. Two Groups (A and B) were treated with 1.25 mg of TPA, 33/week for 20 weeks, and 2 other groups (C and D) were treated with 1.5 mg of TPA, 23/week for 20 weeks. The data depicted represent the maximal papilloma burden achieved by each mouse by the end of the 20-week-exposure period.

Even under the best of conditions, we often see a “cage effect” among multiple-housed mice of the negative control groups or among mice treated with inactive chemicals. It is not unusual to observe, in one of the several (3–5) cages that comprise a dose group, that a dominant female will inflict bite wounds on 2 or 3 of the other cage mates, usually on the lower dorsal area. In another cage of that same dose group, the spontaneous incidence among 4 –5 mice will be zero. Since dominance is established early, the mean latency time to the first woundinduced tumor is not significantly different from that for the positive control group, but the average tumor multiplicity among the multiple-housed, negative control groups has always been less than 1.0. The spontaneous tumor incidence among singly-housed Tg.AC mice reported by other investigators ranges between 4 to 6%. Among the negative control groups in a recent study conducted by the National Toxicology Program (Eastin et al., 1998), only 6/162 (3.7%) male and 8/185 (4.3%) female mice of the singly-housed hemizygous Tg.AC animals, developed papillomas at the site of administration (Mahler et al., 1998). Holden et al. (1998) reported a 2.5% (2/80) incidence of

papilloma bearing animals among negative control singlyhoused hemizygous Tg.AC mice and 8/130 (6.0%) negative control mice developed papillomas in a study reported by Blanchard et al. (1998). Rarely did these mice develop more than a single papilloma. It has been our experience that the skin of the Tg.AC mouse is not unduly sensitive to everyday laboratory hazards such as nicks and abrasions that may be caused during handling, animal care, shaving, and administering treatments. We have been unable to attribute any significant incidence of skin tumors to such injuries, most of which tend to be rather superficial. Rarely, a full-thickness nick caused by the shaver will induce a papilloma at the wound site. The procedure for microchip implantation for identification purposes results in a small fullthickness wound located in the mid-dorsal area. There has been no proven association of papilloma induction occurring at these wound sites (Holden et al., 1998: Blanchard et al., 1998). TPA serves as a sensitive, positive-control agent and its use provides the investigator with the opportunity to gain experience in assessing a positive response. The regular use of a positive control article also permits an assessment of the vari-


ability of responses that may occur from one shipment of animals to another. In the studies cited here, 70/75 homozygous and 47/49 hemizygous mice developed tumors. Papillomas were induced in both hemizygous and homozygous Tg.AC mice as early as 5 weeks under conditions of more aggressive dosing with TPA; e.g., 1.25 mg, 33/week or 1.5 mg, 23/week. The value of conducting a positive control group, utilizing TPA (at doses of 1.25–2.5 mg) was demonstrated in more recent studies conducted in Tg.AC transgenic mice. The use of these groups as a phenotypic monitor led to the recognition of the emergence of a “nonresponder phenotype”. Beginning in 1997, some of the animals in the TPA-positive control groups failed to develop papillomas. These aberrant responses have been reported by Weaver et al., (1998) and Blanchard et al. (1998). Subsequent application of the genotyping procedure described by Thompson et al., (1998) has resulted in the elimination of the nonresponder phenotype from the foundation breeding colony (unpublished observation). With the exception of the study reported in Table 4, survival of mice during the 20-week-exposure periods was similar among negative and positive control groups and the groups treated with the test articles. Survival varied most widely among the groups reported in Table 4. This was primarily due to the fact that the mice were older (18 weeks) at the start of the study, and animals that developed odontogenic tumors near the end of the study had to be removed. Survival can also be effected by audiogenic seizure, a characteristic of the FVB/N mouse strain in which the transgenic model was created. A recent report by Goelz et al., (1998) describes the neuropathologic findings associated with the occurrence of seizures in FVB/N mice. Occasionally, the auditory stimulus of the shaver can induce a fatal seizure. Mice found dead without apparent cause may have died from a seizure. The severity of the seizures is variable and in some instances, the mice can recover and do not require removal from the study. The sporadic neoplasms associated with expression of the v-Ha-ras transgene are not commonly seen in Tg.AC mice until after 6 months-of-age. The most likely sporadic tumor to be observed is an odontogenic tumor of the jaw that may appear in low incidence toward the end of the chemical exposure period. The incidence may be as high as 15% (Mahler et al., 1998) and does not have a significant impact on the interpretation of study results. The incidence of odontomas in homozygous mice can be as high as 37% at one year-of-age (Wright et al., 1995). Their origin appears to be in the periodontal ligament of the incisor tooth buds (Mahler et al., 1998; Wright et al., 1995). Erythrocytic leukemia, salivary gland tumors, and yolk sac carcinomas of the ovary can occur in a range of 1–3 percent incidence (Hansen et al., 1996; Mahler et al., 1998). Well-advanced erythrocytic leukemia is associated with hepatomegaly and splenomegaly, which can be easily observed grossly in live animals; livers weighing up to 10.0 grams have been found at sacrifice (Trempus et al., 1998). Because mice aggressively groom the area of topically applied chemical

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agents, it is not unusual to find papillomas of the forestomach at terminal sacrifice, due to oral exposure. This has been especially noticed in TPA-treated animals. Also, an increase in erythrocytic leukemias has been associated with mice with high tumor burdens induced by TPA. In addition to papillomas, topically applied benzene induces granulocytic leukemia (unpublished results). All spontaneous and induced tumors examined to date have exhibited transgene expression, which has been localized to the proliferative areas of the tumors (Hansen and Tennant, 1994a; Hansen et al. 1996). The incidence of spontaneous tumors in Tg.AC mice does not impact on or confound the interpretation of the chemicallyinduced tumor response in the 20- to 26-week exposure studies because the incidence is so low during this period. In addition, we have not yet seen any chemically related increase in the spontaneous tumors that characterize the wild type FVB/N parent strain (Mahler et al., 1996). The dose selection for the Tg.AC transgenic studies was based on the maximum tolerated dose (MTD) and the dose range selected for the 2-year bioassays. For most of the chemicals, a higher than MTD dose was used as the top dose if survival had not been affected at that dose in the NTP 13-week subchronic dose-finding studies. The animals were examined daily and there was no evidence that the doses selected caused ulceration or overt irritation during the course of the studies. The correlation of activity in the Tg.AC mouse model with the NTP 2-year bioassay results was high. Three of the 6 chemicals, benzene, lauric acid diethanolamine, and o-benzylp-chlorophenol exhibited clear activity in Tg.AC mice and BZ and LADA induced dose-related increases in the papilloma response and reductions in tumor latency times. These results were in agreement with the activity outcomes of the 2-year bioassays. Benzethonium chloride, 2-chloroethanol, and triethanolamine did not induce any activity and the responses for BZTC and 2-CE were also in agreement with the bioassay results. However, the inactivity of TEA, a non-genotoxic carcinogen, was not in agreement with the bioassay result in which the high dose of TEA had increased the incidence of liver tumors only in female mice. In the studies reported here, the relative sensitivities of the hemizygous and homozygous genotypes to TPA. BZ, and LADA, indicated that at similar tumor inducing doses, the average latency for tumor induction will be reduced and the average maximum tumor number will be reached earlier in the groups of homozygous mice. It is important to note, however, that these comparative studies indicate that both Tg.AC genotypes would respond in a convincing manner to an active chemical, when a protocol using 3 or more chemical doses is employed. The reproducibility and sensitivity of the hemizygous female Tg.AC mice to slightly different dosing regimens of TPA, the positive control agent, is instructive. The papilloma response induced by 1.25 mg TPA, 33/week, after 20 weeks of exposure, was not significantly different from that of 1.5 mg TPA, 23/week. Animals receiving the higher total

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weekly dose (3.75 mg) did show a more uniform response, and papillomas were observed at 5 weeks, 2 weeks earlier than in the groups receiving the lower weekly dose. All but one animal had 13 or more papillomas. There was a more variable response among the mice receiving a total weekly dose of 3.0-mg TPA. But among all treated mice, papillomas were induced in 47/49 mice, indicating that hemizygous female Tg.AC mice are nearly as sensitive to a conservative TPA dosing protocol as are homozygous female mice. The 2 statistical analyses used to evaluate the skin tumors were complementary, each dealing with a different aspect of the skin papilloma response. The life table analysis is sensitive to both an increased tumor incidence and a shortened tumor latency, but it does not take tumor multiplicity into account. The Jonckheere and Mann-Whitney U-tests, when applied to maximum tumor yield, assess tumor multiplicity but do not make a distinction as to when the maximum tumor yields occurred. Moreover, if reduced survival is a problem (which was not the case in these studies), then the latter procedures could be applied separately for groups of animals with comparable survival, and the results pooled statistically to obtain an overall result. We are currently working on the development of new statistical methodology that may allow an assessment of tumor incidence, onset time, and multiplicity in a single unified analysis. In these studies, animals were treated for only 20 weeks. We now recommend exposures of 26-week duration. This 30% extension of the dosing period should provide added credence to studies that yield negative results. However, studies yielding unequivocally positive results earlier than at 26 weeks need not be carried for the full experimental period. Although we have usually terminated experiments at the end of the 10-week observation period, in some instances animals were kept up to one year after administration of the first chemical dose, in order to observe progression of benign squamous-cell papillomas to malignancy. In every case, skin malignancies were observed and described as either squamous- or spindle-cell carcinomas. The incidence of progression to malignancy is about 40%, and the malignancies always occur in association with a pre-existing papilloma (Hansen et al., 1995). Dose selection for test articles of unknown toxicity should follow established procedures for determining an MTD. Doses that cause acute or chronic irritation or ulceration should be avoided. The dose-finding studies may be performed in FVB/N wild type mice, since there is no reason to expect that absorption or chemical disposition will be any different in the parent strain than in the Tg.AC mice (Sanders et al., 1998). The results of these studies provide evidence for support for the adjunctive use of the Tg.AC transgenic mouse skin tumor model in the assessment and interpretation of the carcinogenic potential of chemicals. Reduction in time and cost are just 2 of the most important advantages of using the Tg.AC mouse tumor model for assessing chemical activity. The induction of skin tumors by topical application of the test agent equates to

a reporter phenotype that is easily observed and quantified without sacrifice of the animal. The response time to chemical treatment occurs by 26 weeks, which is one-4th of the exposure time currently required in the standard 2-year bioassay. The number of animals (15–20) per sex per dose group is substantially less than the 50 animals per sex/species now used in the standard bioassay. Although the number of chemicals evaluated in the studies reported here are small, over 20 other chemicals have been tested in the Tg.AC mouse model and results for 11 of these were recently reported by (Eastin et al., 1998). There is a high correspondence between the NTP 2-year bioassay results and the chemical activity observed in the Tg.AC transgenic mouse model. Furthermore, 5 human carcinogens, benzene, diethylstilbestrol (DES), cyclosporin A, melphalan, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were all active in Tg.AC mice (Eastin et al., 1998). Three of these, DES, cyclosporin A, and TCDD are non-genotoxic agents. An important characteristic of the Tg.AC model is that, in addition to detecting genotoxic compounds, it is also sensitive to non-genotoxic agents that are believed to induce tumors via epigenetic mechanisms. Our data also indicate that the model is more likely to detect trans-species carcinogens than those that induce tumors only at one site in a single sex/species. Further development of this and other transgenic mouse models holds great potential for facilitating the detection of carcinogenic chemicals and strengthening the interpretation and regulatory utility of chronic rodent toxicity and carcinogenesis studies. The Tg.AC model, along with 2 other transgenic models, the p53 1/– deficient mouse (Donehower et al., 1992; Harvey et al., 1993) and the TgHras2 mouse (Yamamoto et al., 1996, 1998), are currently undergoing extensive evaluation (Robinson, 1998). Over 20 chemicals are being evaluated in these transgenic mouse models by members of the International Life Sciences Institute (ILSI) under the auspices of the Alternatives to Carcinogenicity Testing Technical Committee (Robinson, 1998). The results of these studies will provide important information that will define the limits and usefulness of these transgenic models. ACKNOWLEDGMENTS We would like to thank Tadesse Woldetsadik, ILS, for performing the animal treatments, Kathryn Babson for typing the manuscript, and Stanley Stasiewicz for editing/formatting the tables and figures in a creative manner and for his perseverance in directing the manuscript to its final form.

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