Spontaneous tumors in SENCAR mice.

A total of 305 female and 36 male SENCAR mice in six different groups were observed for 540 to 875 days for tumors as well as nonneoplastic lesions. Three of these groups were observed for their lifespans. The 123 females and 36 males, observed for their lifespans (800 to 875 days) showed median survival times ranging from 730 to 745 days. The average peak body weights were 42.4 g for females and 50.0 g for males. In the animals observed for their lifespans, the total tumor incidence was 65.6% for females and 69.4% for males. Both sexes showed a high incidence of papillary tumors of the lung, 16 to 39%, which was independent of aging. Females showed a 5.1 to 21.0% incidence of mammary tumors, which increased with age. Males showed a high incidence of hyperplastic nodules of the liver, i.e., 12 of 36 animals. Other frequently occurring tumors were leukemia and papillomas of the forestomach.


Introduction
In a companion report from this laboratory (1), we described the use of female SENCAR mice in the bioassay of chemicals for cocarcinogenic and tumor-promoting activity and, in some instances, compared the skin tumor responses in these animals with those observed in our earlier work with female ICR/Ha mice (2).
During the course of the above work, and including earlier trial experiments with SENCAR mice (3), we have accumulated some background information on the incidence of spontaneous tumors (benign and malignant) and nonneoplastic lesions in SENCAR mice. In these no-treatment control groups, 305 females and 36 males were observed in six different experiments for periods ranging from 540 to 875 days.
Because of the current interest in the use of these mice for skin carcinogenesis assays, which was the main topic of the conference at which the above paper (1) was presented, it was considered useful to present our findings on the incidence of spontaneous lesions in SENCAR mice.
The findings reported here were accumulated during the course of mouse skin cocarcinogenesis research.
Hence, we do not have the detailed results that would have been produced from a protocol designed for the purpose of studying spontaneous tumors in SENCAR mice. "Mice were kept to end of lifespan.

Methods
All mice were obtained from Oak Ridge National Laboratories (Oak Ridge, TN). They were used as no-treatment control groups for concurrently running skin cocarcinogenesis and two-stage experiments, some of which have been reported earlier (1,3). The six groups of mice represent animals used over a period of 7 years in this laboratory. They were 4 to 7 weeks of age at time of arrival in the laboratory. Animals were allocated randomly and numbered by toe-clipping during the week of arrival. After one week, they were assigned to experimental groups as no-treatment controls. Mice were housed, five per stainless steel cage, on heattreated hardwood chip bedding. Males and females were housed in separate animal rooms, and males showing aggressive behavior were separated and housed individually. Rooms were maintained at 68 to 72°F and 50% relative humidity. Fluorescent lighting used was on a 12-hr on-off cycle, and fresh air was supplied at the rate of 12 to 15 changes per hour. The animals had free access to Purina Rodent Chow (Number 5001) and tap water.
The six groups and relevant information are given in Table 1. Groups 1 to 3 were matched with skin application groups and hence were killed at the termination of the experiment. Groups 4 to 6 were kept to the end of their lifespans.
Animals in moribund condition were killed. Necropsies did not include the cranium, and routine sections of tissues were not taken for groups 1 to 3. Only those tissues which appeared abnormal by gross examination were taken for histopathology. In groups 4 to 6, routine sections of the following clinically normal tissues were taken in addition to abnormal tissues: lung, liver, kidney, stomach, spleen, and skin. Tissues were fixed in formalin and stained with hematoxylin and eosin; special stains were not used.

Survival
The survival curves for groups 4, 5, and 6, which were allowed to live out their lifespans, are shown in Figure  1. The median survival times for these three groups are shown in Table 1.

Body Weight
Body weights of the six groups of animals are shown in Figure 2. The average peak body weight was 42.4 g )0 600 700 800 900 950 for females, and peak body weight for the one group of males was 50.0 g.

Tumors
Types of tumors observed are presented in Table 2, along with the number and percentage of mice in each group with each type of tumor. The mammary tumors were defined by several criteria. Tumors were classified as fibroadenomas if proliferation of both the epithelium and stroma was present and no necrosis was observed. If necrosis was present, then the tumors were designated as adenocarcinomas. If the tumor could not be classified as either a fibroadenoma or an adenocarcinoma, the generic term mammary tumor was used. These latter tumors are designated as unclassified tumors in Table 2.

Nonneoplastic Lesions
Nonneoplastic lesions were seen in all six groups of SENCAR mice examined and are listed in Table 3. With the few exceptions noted below, these lesions were all clinically apparent at necropsy and thus represent a conservative number. In the males, group 5, cystic dilatation of the seminal vesicles was probably an agerelated lesion. The changes seen in the male skin and some of the inflammatory changes seen in the kidneys may have been attributable to amyloidosis. We did not use special stains for amyloid, since nonneoplastic lesions were not of prime interest in our study.

Routine Sections
The numbers of routine sections of various organs taken from groups 4 to 6, and the corresponding diagnoses are shown in Table 4. The total number of clinically normal sections from all tissues listed in Table 4 was 143. Of these, only 10, i.e., about 7%, were diagnosed as tumors or nonneoplastic lesions. Thus, these routine sections did not markedly alter the overall tumor and nonneoplastic lesions incidences shown in Tables 2 and 3.

Discussion
An examination of the tumor incidences in SENCAR mice used in this laboratory, listed in Table 2, revealed that five tumor types occur most frequently. These tumor types and the frequency of their occurrence are summarized in Table 5. These tumors occurred in one or more of the six groups with a frequency of above 10%. With the exception of group 3, the overall tumor inidences were greater than 50%. The most commonly 50. occurring lung tumors were benign papillary tumors, and the frequency of incidence ranged between 16 and 39% in the six groups. The incidence of lung tumors does not appear to be age related, e.g., group 1, which was observed for 540 days, had essentially the same lung tumor incidence as that of groups 5 and 6, which were observed for 815 and 875 days, respectively. In another study (4), the most frequently observed tumors were those of the lung in CD-1 mice of both sexes. Male mice showed a high incidence of hyperplastic nodules of the liver (group 5), 33.3%, as compared to females, which showed a range of 0 to 2.4%. This difference between sexes has also been observed in B6C3F1 hybrids and other mouse strains (5).
The mammary tumor incidences summarized in Table  5 showed a distinct increase with age, from 5.1% in group 1 (540 days) to 22% in group 6 (875 days).
It was of interest to compare median survival times and body weights of SENCAR mice from some of the     control groups discussed here with ICR/Ha mice used in most of our earlier studies on promoters, cocarcinogens, and whole carcinogens. Groups 5 and 6 from the present report were compared with ICR/Ha mice of both sexes used as control groups in an earlier study (6). This comparison is illustrated in Figure 3. The markedly higher weight and increased median survival times of SENCAR mice compared to ICR/Ha mice of both sexes are noteworthy.
In the course of the preparation of this report and another one for the symposium (1), the differences in results obtained in different laboratories with the same strain of mice became obvious. The fact that these differences are apparent stressed to us again the importance of standardizing laboratory animal care and han- dling procedures. So many variables, often overlooked and thought of as having minor relevance, are in fact important for comparing chronic animal bioassay results among different laboratories. The importance of all these variables has been discussed and reviewed (7).
From the papers presented at this symposium, it was clear that the SENCAR mouse will be valuable for mouse skin tumorigenesis studies, particularly in twostage, i.e., initiation-promotion experiments. In this regard, the utility of SENCAR mice for carcinogenesis studies relating to tissues and organs other than skin merits further attention. This point is important because in vitro models, while most valuable, must continue to be regarded as adjuncts to, rather than replacements for in vivo animal bioassays. The latter view is shared by other researchers in toxicology (8,9).
Our findings from the present work and from that presented elsewhere in this volume (1) indicate the need for further work on the utility of the SENCAR mouse in toxicology research.