Growth, Body Weight, Survival, and Tumor Trends in F344/N Rats During an Eleven-Year Period"

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1 TOXICOLOGIC PATHOLOGY ISSN:O Copyright 1990 by the Society of Toxicologic Pathologists Volume 18, Number 1 (Part 1), 1990 Printed in U.S.A. Growth, Body Weight, Survival, and Tumor Trends in F344/N Rats During an Eleven-Year Period" GHANTA N. RAO,l JOSEPH K. HASEMAN,2 SONDRA GRUMBEIN,3 DENISE D. CRAWFORD,2 AND SCOT L. EUSTIs l 'Naiional Toxicology Program, Division of Toxicology Research and Testing; -Division ofbiometry annd Risk Assessment, National Institute ofenvironmental Health Sciences, Research Triangle Park, North Carolina; and 3Pathology Associates, Inc., Durham, North Carolina ABSTRACT Time trends for growth, body weight, survival and tumor prevalences in 144 diet control groups with a total of 5,184 male F344/N rats and 146 diet control groups with a total of 5,289 female rats ofnci-ntp 2-yr chemical carcinogenicity studies started during an ll-yr period (1971 to 1981) in 11 toxicology testing laboratories were evaluated. Male and female rats in more recent studies grew faster and attained a higher body weight than rats from earlier studies. Survival of males showed a significantly decreasing trend over time, which may have been related to diseases associated with increasing body weight, prevalence ofleukemia and changes in criteria for euthanasia of moribund animals. The time trend for survival of females was not significant. There were highly significant(p < 0.001) positive time trends for prevalences ofleukemia, anterior pituitary tumors and thyroid C-cell tumors in both sexes, adrenal pheochromocytomas in males and mammary tumors and endometrial stromal polyps in females. The prevalence of mammary tumors in females and pituitary tumors in males had a highly significant (p < 0.01) positive association with body weight. Histological reevaluation of tumor prevalences in approximately 250 rats of each sex at each of 4 different time periods indicated that changes in diagnostic criteria may have contributed to but could not totally explain the increased prevalence ofleukemia. Changes in diagnostic criteria and the amount of tissue examined may have contributed to the increased prevalence of anterior pituitary tumors in both sexes and adrenal pheochromocytomas in males. Interlaboratory variability and changes in diet may also have contributed to the time-related trends. Keywords. Time trends; carcinogenicity studies; leukemia; mammary tumors; pituitary tumors; adrenal pheochromocytomas; thryoid C-cell tumors; endometrial polyps INTRODUCTION Small rodents-especially rats and mice-are recommended for chemical carcinogenicity studies (5, 10). Major reasons for selection ofsmall rodents are their short life span and small size. They can be conveniently housed in large numbers in a controlled laboratory environment, thus, providing a sufficient number of animals for statistical evaluation of results. The National Toxicology Program (NTP) of the National Institute of Environmental Health Sciences, previously the Carcinogenesis Bioassay Program of the National Cancer Institute (NCI), has evaluated chemicals for toxicity and carcinogenicity in rats and mice since In recent * Address correspondence to: Dr. G. N. Rao, NIEHS/NTP, MD AG-GI, P.O. Box 12233, Research Triangle Park, North Carolina years, there have been reports (15) and commentaries (16, 17, 21) suggestingthat rodents (especially rats) became obese in carcinogenicity studies (16, 21) and that there is an epidemic oftumors in those rodents (16). Becauseofchanges in selection, disease control, improved diet, and better control of environmental conditions, there has been a substantial increase in body weights (16) and life span (10) of rodents used in long-term toxicity studies during the last 2 decades. Yearly survival patterns and 7-yrcumulativegrowth curves for untreated and gavage vehicle control groups off344 rats of the NCI carcinogenicity studies started between 1971 and 1977 were reported by Cameron et al (3). The purpose of this report is to review and evaluate time trends for growth, body weight, survival, and tumor prevalences in F344/N rats of the NCI-NTP studies started between 1971 and 1981.

2 62 RAOETAL TOXICOLOGIC PATHOLOGY Animals MATERIALS AND METHODS The Fischer 344 (F344/N) inbred has been the selected rat strain for the NCI-NTP studies since 1971 (3,24). The source ofrats from 1971 to 1976 was described by Cameron et al (3). Since 1977, the rats were produced in pathogen-free ("strict barrier") production colonies from foundation colony breeding stock obtained from NIH genetic colonies. The F344/N rats used in carcinogenicitystudies were the progeny of pedigreed breeders that were transferred from the production colonies to testing facilities when they were 4-5 weeks ofage. Diet In studies started between 1971 and 1979, the rats were fed commerical closed formula (2) diets.v> The NIH-07 open formula (2) rat and mouse ration with approximately 24% crude protein (2, 12) was the selecteddiet for studies started in 1980 and Carcinogenicity Studies After quarantine for 2-3 weeks at testing laboratories, rats were distributed into experimental groups of 50 per sex. Some studies started during the years had 15 to 30 rats per group. Rats in all studies were housed 4-5 per cage by sex in plastic cages with solid bottoms and sides. Each study had one untreated or diet control group and 2 or more treatment groups. The chemical to be evaluated was administered by incorporation in the diet. This report includes diet control groups of144 2-yr chemical carcinogenicity studies for a total of 5,184 control male rats and 146 studies for a total of5,289 control female rats started during the period 1971 to These studies were conducted in 11 different toxicology testing laboratories located throughout the United States and followed standardized NCI-NTP experimental procedures. Study distribution by year and testing laboratory is listed in Table 1. Due to experimental variables related to route of administration, more than 150 studies started during this time period involving gavage, skin painting, and inhalation methods of chemical administration were not included in this investigation. Animal use and care for the NCI-NTP studies were in accordance with the United States Public Health Service policy on humane care and use of animals and the Guide for the Care and Use oflaboratory Animals (11). 4 Purina Rodent Chow, Ralston Purina Company, St. Louis, Missouri. s Wayne Lab-Blox, Continental Grain Company, Chicago, Illinois. Growth and Body Weight Rats were weighed at 1-4-week intervals throughout the course of a study. Growth patterns were determined by computing 9-week moving averages for the body weight values ofall diet control groups started during a given year. Separate growth curves were calculated for each of the eleven years examined. For assessing interstudy variability in body weight, the measure chosen was the maximum of the mean body weights for the control group observed during a study. Male rats attain a maximum body weight at 80.2 ± 9.6 (mean ± SD) weeks of age, while the corresponding value for females occurs at 103 ± 7.3 weeks of age (7). Survival The number of rats surviving to 106 weeks ofage (approximately 100th week or 23rd month of the study) for the diet control groups of each study was determined and an average 106-week survival rate with standard deviation for the studies started during each calendar year was computed. Tumor Prevalences Rats that died during the course of a study were necropsied. Rats observed to be in moribund condition and rats surviving to the end of a study (2 yr) were euthanatized and necropsied. At necropsy, samples of20-40 tissues and grossly observable tumors from each rat as required by protocol were fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned at 5 JJ.m. Sections stained with hematoxylin and eosin (H&E) were evaluated for neoplastic lesions by light microscopy. Time trends for common spontaneous tumors with a prevalence ofapproximately 10% or more were determined from the historical database of the NCI NTP studies. These tumors included leukemia (hematopoietic system tumors), anterior pituitary tumors, adrenal pheochromocytomas and thyroid C-cell tumors in males and anterior pituitary tumors, mammary gland tumors, leukemia, thyroid C-cell tumors, and endometrial stromal polyps in females. Since almost all male rats may have interstitial cell tumor of the testes (7), a time trend for this tumor was not evaluated. Reevaluation oftumor Prevalences Prevalences of the selected tumors in the NCI NTP historical database were influenced by the experimental procedures, diagnostic criteria and other factors applicable to the time periods when they were evaluated. Some of these factors may have varied from year to year and changes in diagnostic criteria over time or differences between patholo-

3 Vol. 18, No.1 (Part 1),1990 BODY WEIGHT, SURVIVAL AND TUMOR TRENDS IN RATS 63 TABLE I.-Number of studies by year and testing laboratory included in this investigation. No. of studies No. ofstudies Labo- Year" Male Female ratory Male Female I IS I I 1979 II II II 3 3 Total a Year ofstudy start. Studies ended 2 yrlater (e.g., 1971 starts ended in 1973, 1980 starts in 1982). gists (1) may have contributed to changes in the prevalence of some tumors. In an attempt to understand the influence of these factors, especially diagnostic criteriafor identification oftumors, H&E stained tissue sections from the NTP Archives of approximately 250 rats of each sex from all the rats ofdiet control groups in five or more representative studies (some studies started during the period had only rats/group) during each of the time periods , , , and were histologically reevaluated by one ofus (SG), using the same diagnostic criteria for all the time periods. Reevaluation included approximately 1,000 rats of each sex (-19% of the total sample) for the prevalences of leukemia, anterior pituitary tumors and thyroid C-cell tumors in both sexes and adrenal pheochromocytomas in males. During reevaluation, the tissue area of the largest section of each tissue (e.g., pituitary, thyroid, adrenal medulla) of each animal available for microscopic examination was also determined to evaluate the relationship, ifany, between the amount of tissue and tumor prevalences. MO al zee i 200 '50 '00 see '60 : : 400 g see al : I MALE RATS Legend a x ro M AGE INWEEKS FEMALE RATS Legend lo- o I I x o 1'0 AGE INWEEKS FIG. 1.-Growth curves for male and female F344 rats of diet control groups started in 1972, 1978, and RESULTS Growth and Body Weight Growth curves for diet control groups of male and female rats started during the years 1972, 1978, and TABLE n.-survival to 106 weeks of age for male and female F344/N rats. Statistical Procedures Multiple regression procedures utilizing the GLM procedure ofsas (19) were employed to assess the significanceof time-related trends for survival, maximum mean body weight and tumor prevalences, after adjusting for inter-laboratory variability. Additional regression analyses (20) were carried out to evaluate the association between tumor prevalences and body weight as well as survival. The basic experimental unit in these analyses was the study, i.e., n = 144 for male rats and n = 146 for female rats (Table I). Differences in diagnoses between the original and reviewing pathologists were evaluated by a sign test (20). Year" II-yr average Significance of time trend a Year of study start. 'Mean. c Standard deviation. Male 85.3' (17.5Y 76.7 (15.8) 81.6 (10.9) 77.0 (7.8) 65.5 (10.9) 74.1 (9.4) 74.5 (7.5) 75.0 (6.6) 75.7 (7.4) 66.4 (7.5) 62.5 (8.5) 74.0 p < Survival (%) Female 83.3 (12.6) 77.9 (15.5) 82.3 (13.4) 83.3 (7.7) 81.0 (8.7) 81.8 (6.4) 78.7(6.8) 78.8 (5.9) 84.4 (5.7) 75.0 (8.9) 70.8 (7.8) 79.8 p = 0.082

4 64 RAOET AL TOXICOWGIC PATHOLOGY 1981 are presented in Fig. 1. These curves are representative of the general patterns of growth observed in these studies and reflect 9-week moving averages. In more recent studies, male and female rats grew faster and attained a higher maximum body weight than in the earlier studies. Averages of the maximum mean body weights for studies started during each of the 11 yr are given in Fig. 2. Males showed a significantly (p < 0.001) increasing body weight trend from 1971 to 1981 with an average maximum mean body weight of408 gin 1971 study starts to 480 g in 1981 study starts (Fig. 2). Females also showed a significantly increasing (p < 0.001) body weight trend of307 g in 1971 to 349 gin 1981 starts. Interlaboratory variability (8) was significant (p < 0.01). Time trends were highly significant (p < 0.001) even after adjusting for interlaboratory variability for both sexes. Survival Mean 106-week survival rates with standard deviation of male and female rats for each of the 11 yr are listed in Table II. There were fluctuations in survival from year to year with a mean of 74.0% (range of 62.5 to 85.3%) for males and a mean of 79.8% (range of 70.8 to 84.4%) for females. Statistical evaluation indicated highly significant (p < 0.001) decreasing time trend for survival of male rats, after adjusting for interlaboratory variability, which was also significant (p < 0.01). Decreased survival was also associated with elevated body weight. The interlaboratory variability and the time trend for survival were not significant for female rats. Tumor Trends Trends for prevalences of hematopoietic system tumors (mostly mononuclear cell leukemia) in male and female rats are presented in Fig. 3. There were highly significant(p < 0.001) increasing trends from 1971 to 1981 study starts for both sexes. The prevalence of leukemia in control male rats was low (7.9%)in 1971 starts and high (48.0%)in 1981 starts. The prevalence of leukemia in control female rats also was very low (2.1%) in 1971 and high,(24.6%) in 1981 starts. In both sexes, the interlaboratory variability was highly significant (P < 0.001). Association ofbody weight with prevalence ofleukemia was not significant when adjusted for interlaboratory variability. Prevalence of leukemia was inversely associated with survival in both sexes indicating that leukemia was generally a fatal disease. Prevalences of anterior pituitary tumors with time trends for male and female rats are shown in Fig. 4. The time trends were highly significant(p < 0.001) with increasing prevalences for both sexes. How- >20 : :lao :lao 310 s 340 g MALE RATS FEMALE RATS P < e ", '!1 'o ".f' q,.. <f' q,' YEAR P < 0.001,q;.' ", '!1..,cr;.'" ".f' q,.. <f' q,' I YEAR FIG. 2.-Maximum mean body weight by year of study start and time trends for body weight ofmale and female F344 rats ofdiet control groups from 1971 to 1981 (mean ± SD). Least squares regression line is given. Body weight trend shows significant (P < ) linear increase over time. ever, in males there were wide fluctuations from year to year with high prevalence (34.7%) in 1979 and low prevalence (10.3%) in 1972 starts. In females, the increase in prevalence was gradual with a low (26.5%) in 1971 and a high (50.8%) in 1979 starts. Interlaboratory variability in the prevalences ofanterior pituitary tumors was highly significant (p < 0.00 I) in both sexes. The occurrence of pituitary tumors showed a significantpositive association with body weight for males but a similar association in females was not statistically significant. There was no significant association between survival and prevalence ofanterior pituitary tumors in either sex. Prevalences of thyroid C-ce11 tumors by year of study starts for male and female rats are listed in Table III. The time trends were highly significant (P < 0.001) for both sexes. There was a gradual increase in tumor prevalences over time with marked

5 Vol. 18, No.1 (Part 1),1990 BODY WEIGHT, SURVIVAL AND TUMOR TRENDS IN RATS 65 MALE RATS MALE RATS P < g > o P < ' # '" 'o.,..(' 'I> -.,-.'1>0.(6'1>' YEAR FEMALE RATS s ' # '" 'o.,..(' 'b...(6'b' YEA" FEMALE RATS 40 >- :1 P < '... '" 'o.,..(' 'I>...(6tf>.(6'b' YEAR FIG. 3.-Prevalences by year of study start and time trends for hematopoietic system tumors (mostly leukemia) ofmale and female F344 rats of diet control groups from 1971 to 1981 (mean ± SD). Least squares regression line is given. Tumor trend shows significant (p < 0.001) linear increase over time. l---.:''''-#":'' ,,:r.:-$---=/l);t"'-$---="':'"""'-.(''-$--=.,.r-,-$--=,,,r-.. -(6tf'"="-.(6'1>":T YEAR FIG. 4.-Prevalences by year of study start and time trends for anterior pituitary tumors of male and female F344 rats of diet control groups from 1971 to 1981 (mean ± SD). Least squares regression line is given. Tumor trend shows significant (p < 0.001) linear increase over time. TABLE III. - Prevalences of adrenal pheochromocytomas and thyroid C-cell tumors in the male and thyroid C-cell tumors, uterine stromal polyps and mammary gland tumors in female F344 rats. Thyroid C-cell tumors (%) Adrenal pheochromo- Uterine stromal Mammary gland Year cytornas (%) male Male Female polyps (%) tumors (%) female " (4.7)b 8.4" (3.0)b 3.4" (3.1)b 14.2" (16.7)b 21.7" (5.8)b (6.3) 7.4 (8.3) 5.6 (4.8) 11.6 (11.1) 17.3 (9.4) (6.5) 8.5 (6.9) 8.6 (7.7) 13.5 (9.6) 20.9 (11.2) (6.5) 7.5 (8.8) 5.5 (2.7) 12.3 (13.1) 17.1 (10.4) (7.5) 5.1 (4.2) 6.2 (5.1) 16.2 (5.9) 26.5 (9.6) (7.9) 9.0 (4.7) 6.4 (3.0) 20.1 (7.4) 23.6 (7.2) (10.4) 8.4 (4.5) 9.9 (6.2) 24.6 (7.2) 28.0 (8.9) (5.8) 10.5 (4.3) 9.6 (5.7) 16.5 (6.4) 25.2 (10.1) (10.0) 11.2 (6.0) 9.7 (4.9) 16.6 (7.1) 25.2 (14.8) (14.1) 15.1 (8.2) 15.3 (6.9) 18.9 (9.6) 33.6 (13.1) (11.0) 14.9 (5.8) 14.8 (9.9) 19.6 (11.9) 35.0 (13.3) p < p < p < p < p < "Mean. Standard deviation. c Significance of time trend.

6 66 RAOET AL TOXICOLOGIC PATHOLOGY TABLE IV.-Original and reevaluated prevalences for leukemia, anterior pituitary tumors, and thyroid C-cell tumors in male and female and adrenal pheochromocytomas in male F344 rats for the four selected time periods. Anterior Thyroid Adrenal pituitary C-cell pheochromostudy animals Year of Number of Leukemia (%) tumors (%) tumors (%) cytomas (%) starts reevaluated 0 R 0 R 0 R 0 R Male Statistical significance- p < NS p < p < Female Statistical significance- p < p < p < a = Significanceof differences between the reviewing pathologist diagnoses and the original pathologist diagnoses for the 4 selected time periods; NS = not significant. b = Prevalence of this tumor in the female rats was less than 10%; therefore, trends were not evaluated. o Original; R = Reevaluation. increases in 1980 and 1981 study starts. Interlaboratory variability was not significant and the prevalence of thyroid C-cell tumors was not associated with body weight or survival in either sex. Prevalences for adrenal pheochromocytomas in male rats are listed in Table III. The rates showed a highly significant (P < 0.001) positive time trend and increased from 8.0% in 1971 to 38.1% in 1980 starts. There was a marked increase in the prevalence from 1979 to Interlaboratory variability was not highly significant (p > 0.01) and there was no significant association with body weight and survival. The prevalence of adrenal pheochromocytomas in female rats was less than 10%; therefore, time trend for this tumor was not evaluated. The prevalences of endometrial stromal polyps are presented in Table III. There were wide fluctuations from year to year with a low (11.6%) in 1972 to a high (24.6%) in 1977 starts. Statistical evaluations indicated that there was a highly significant (p < 0.001) positive time trend. Interlaboratory variability was highly significant and there was no significant association with body weight or survival. The prevalences of mammary tumors with time trend for female rats are presented in Table III. There was a highly significant (p < 0.001) but gradual increase in the rate of mammary tumors over the l l-yr period. There were wide fluctuations between years and prevalences ranged from 17.1% in 1974 to 35.0% in 1981 starts. Interlaboratory variability was highly significant(p < 0.001). There was a highly significant (P < 0.001) positive association between body weight and mammary tumor prevalences and no significant association between mammary tumor prevalences and survival. Reevaluation oftumor Prevalences Prevalences of leukemia, anterior pituitary and thyroid C-cell tumors in both sexes and adrenal pheochromocytomas in males as determined by histological reevaluation using standardized diagnostic criteria along with prevalences from the original evaluations for the same 250 male and female rats at each of4 selected time intervals are listed in Table IV. While there were significant differences in the diagnoses ofthe original and reviewing pathologists (Table IV), in most cases these differences were relatively consistent across time intervals, and thus, could not explain all the time-related increases in tumor prevalences. For both male and female rats, the reevaluation revealed a higher prevalence ofleukemia in all time groups than previously diagnosed, reflecting an increasing recognition of early stages ofleukemia. For females, differencesin diagnostic criteriamight have been partly responsible for the apparent time-related trend in this tumor (see Table IV). However, it is clear that these time-related trends for both sexes cannot be explained solely on the basis ofdifferences in diagnostic criteria. For male rats, the prevalence ofanterior pituitary tumors on reevaluation was similar to that originally reported. For females, consistently fewer pituitary tumors were diagnosed on reevaluation, particularly in the more recent studies, and thus, the significance ofthe time-related trend was reduced. Thus, changes in diagnostic criteria over time may have been an

7 Vol. 18, No.1 (Part 1),1990 BODY WEIGHT, SURVIVAL AND TUMOR TRENDS IN RATS 67 TABLE Y.-Relationship between size of pituitary, thyroid, and adrenal medulla section examined and tumor prevalences for control male F344 rats Studies Studies Sections with Sections with no tumors Sections with tumors no tumors Sections with tumors Size of section (mm') Number %a Number %a %' Number O/oa Number 0/0" %' Pituitary > Total Median size (mm-) Thyroid > I Total Median size (rnrn-) Adrenal medulla > II Total Median size (mm-) a = % of total (214 and 243 for pituitary, 210 and 245 for thyroid, and 222 and 238 for adrenal medulla) sections. = % of sections within the same size grouping [e.g., in studies for pituitary, 5 of the 33 sections (15.2%) in 8.1 to 12.0 mm' group and 6 of 16 (37.5%) in 12.1 to 24.0 mm' group had tumors]. important factor in the apparent time-related increase in pituitary tumors in female rats. For both male and female rats, the reevaluation revealed a higher prevalence of thyroid C-cell tumors than originally reported in all groups, but the time-related increase in these tumors was still evident. For male rats, the prevalence of adrenal pheochromocytomas was much higher in the reevaluation than originally reported, particularly for the earlier studies. For example, for the studies, tumor prevalence was originally reported as only 11%, but was 27% in the reevaluation (Table IV). In contrast, changes in tumor prevalences in more recent studies were relatively small (e.g., from 31% to 38% in the studies). Thus, for this tumor the significance of the time-related trend in male rats was due in part to differences in diagnostic criteria. The amount of tissue and tumor incidences for the pituitary, thyroid, and adrenal medulla sections of the male rats in the reevaluated and studies are listed in Table V. In the studies, the reevaluated anterior pituitary tumor prevalence was only 9% compared with 26% in the studies. However, the median tissue size was only 5.0 mm- in the earlier studies compared with 9.0 mm- in the more recent studies. There was also a clear association between the amount of tissue examined and tumor prevalence. For example, tumors were found in all tissue samples of24 mm- or greater. However, there were only 2 such samples in the early studies compared with 17 in the more recent studies. Even for smaller tissue sizes there was a clear correlation between the amount of tissue examined and tumor prevalence, and if early and later studies were matched by amount of tissue examined (Table V), there was no longer a significant difference in tumor prevalence between these 2 groups. Similar conclusions were made for thyroid tumors and adrenal pheochromocytomas of male rats and pituitary and thyroid tumors offemale rats. DISCUSSION Body weight determinations were not done at exactly the same week for all studies and there may have been fluctuations from one weighingto the next due to experimental variables. Accordingly, it was not possible to obtain mean body weight for all studies started in a year at predetermined weeks. To obtain the best averages for growth curves 5-, 7-, 9-, and l l-week moving averages were computed and

8 68 RAOET AL TOXICOLOGIC PATHOLOGY plotted. The 9-week moving averages were selected for the growth curves presented in Fig. 1 because each average represented at least 2 weights for each control group. Growth curves for each ofthe 11 yr yielded some superimposing or merging curves and so growth curves for 3 representative years with a good number (8-22) of study starts were selected and plotted in Fig. 1. The faster growth and higher body weight (Fig. 1) over the years may be due to (a) control of environmental conditions, (b) improved diet that is not deficient in essential nutrients but may be high in growth enhancing nutrients-protein and fat, and (c) possible inadvertent selection of fast growing, high body weight offspringas parents for future generations. Survival of control rats at the end ofa 104-week study may vary between studies and between testing laboratories due to differences in criteria for selection of moribund animals. Differences in criteria for moribund sacrifice of rats during the last month of a study, when aged animals may have appeared sick, may have decreased the survival at the end of a study. To decrease the influence of such variables, survival at the 100th week or 23rd month of a study (approximately 106 weeks of age) was selected for evaluation of time trends. Lower survival of male rats in the 1975, 1980, and 1981 starts and female rats in the 1981 starts (Table II) may be related to high maximum body weight (Fig. 2). High body weight which may be due to over nutrition and subtle genetic changes has been reported to decrease life span (22). In addition, in 1982 in the interest of humane care of sick animals and to obtain good tissues for histopathologic evaluation, the NTP testing laboratories were advised to follow vigorous moribund sacrifice procedures during the last 3 months of a study. This procedure may have contributed to decreased survival ofrats in studies started in and ended in Furthermore, the NIH-07 diet used for the studies started in may be high in biologically available protein (24%), which may have increased the severity of nephropathy (6) and contributed to the decreased survival of male rats. Decreased survival in recent studies may also have been related to the increased prevalence ofleukemia observed in these studies. Prevalences of leukemia in both sexes showed highlysignificant positive trends (Fig. 3).There were step-wise increases in 1975 and 1980 starts, especially for male rats. The increase in 1980 may be due in part to an NTP conference on tumors ofthe hematopoietic system providing better understanding and defined criteria for diagnosis of this tumor by pathologists evaluating tissues ofthe NTP studies (NTP unpublished data). While changes in diagnostic criteria appeared to have increased the prevalence of leukemia, there is still evidence of a time-related trend based on the reevaluated tumor prevalences (Table IV). Endocrine tumors-especially anterior pituitary tumors-are reported to be associated with body weight (9, 13,22). The increasing prevalence ofanterior pituitary tumors in F344 rats, especially in males, may in part be due to increasing body weight over the ll-yr period (Fig. 2). Reevaluation of the prevalence at the selected periods indicated that changes in diagnostic criteria, particularly in females, and the amount of tissue examined may also have contributed to the increasing time trends for this tumor. The prevalences ofthyroid C-cell tumors showed highly significant positive time trends in both sexes. Reevaluation ofprevalences at the four selected periods did not change the rates or the time trend. Interlaboratoryvariability and association with body weight or survival were not significant, indicating that there may be other factors influencing the prevalence ofthis tumor. Changes in the amount oftissue examined may have contributed to the time-related trend. Further, there was a marked increase in thyroid C-cell tumors in both sexes for the studies started in 1980 and With 1980 starts, the diet was changed from commercial closed formula diets to NIH-07 open formula diet. The NIH-07 open formula diet may be high in biologically available protein (24%), which may have been a factor for high prevalence of thyroid C-cell tumors in studies started in Protein concentration at levels present in NIH-07 diet has been reported to increase the incidence of thyroid tumors in rats (18). Adrenal pheochromocytomas in male rats showed significant positive time trend mainly due to low prevalences in the early 1970s and high prevalences in starts. Interlaboratory variability and association with body weight or survival were not significant. Reevaluation substantially increased the prevalences in studies started during the period (Table IV), indicating that change in diagnostic criteria may be one of the major factors for the increasing prevalences. Changes in the amount of tissue examined may also have influenced the prevalence over time. Prevalence ofpituitary, thyroid, and adrenal medulla tumors was positively associated with the amount of tissue examined (Table V). Further, differences in the amount of tissue examined could explain the time-related trend for these tumors. However, this result is difficult to interpret because the presence of a tumor in a tissue will likely result in a larger section for examination. Thus, it is not clear if more tumors were detected because of the

9 Vol. 18, No.1 (Part 1),1990 BODY WEIGHT, SURVIVAL AND TUMOR TRENDS IN RATS 69 increased amount of tissue examined, or alternatively, if the increased prevalence of tumors resulted in larger sections for examination. Mammary tumor prevalences of female rats (Fig. 5) showed a highly significant body weight associated increasing trend, indicating that the increasing body weight (Fig. 2) may be the major reason for the increasing time trend of this tumor. Weight of the mammary tumor itself may have added to the weight of the female rat. However, body weight associated positive trend in mammary tumor prevalences was reported in female rats (8, 9) as well as in women (4)and the trends observed in the current investigation are in agreement with the literature reports. Mammary tumors are detected by gross appearance at necropsy and confirmed by histologic examination. They are rarely detected by histologic examination alone and therefore prevalences ofthis tumor were not reevaluated. Even though endometrial stromal polyps showed a significant increasing time trend, prevalences of this tumor were high only in and starts. This tumor is detected by gross appearance at necropsy and confirmed by histologic examination. It is rarely detected by histologic examination alone and so prevalences of this tumor were not reevaluated. Time trends reported in this investigation are probably due to a combination of genetic and experimental variables within a laboratory and between laboratories over time. Interlaboratory variability may be mostly due to differences in the experimental procedures between laboratories. Even though F344/N is an inbred rat, there was a difference of at least 30 generations between the F344/N rats used in 1971 study starts and 1981 study starts. Genetic changes (mutations) are part ofthe normal evolutionary process in biologic systems and there may be subtle genetic changes in the F344/N rat over the 30 generations affecting growth and tumor prevalences. Potential sources of variability in rodent carcinogenesis studies within a laboratory, between laboratories and over time include (a) body weight, age, survival and care of animals, (b) sampling and evaluation procedures in pathology, (c) genetic factors, and (d) random factors. Contribution of these factors for variability in tumor rates were reviewed and discussed elsewhere (8). Mononuclear cell leukemia is a diffuse tumor and so it is difficult to establish quantitative criteria for diagnosis of leukemia from histologic sections, especially for the initial stages. A better understanding ofthis lesion by the pathologists in recent years may have contributed to the increasing prevalence over time. Except for testicular and mammary tumors, all other common (> 10%) spontaneous tumors of the F344 rat are solid tumors of small endocrine organs such as the pituitary, adrenals, and thyroid. These endocrine organs are small and often weigh less than 0.1 gram. Due to small size, these tissues are difficult to collect, process, and evaluate for pathologic changes, especiallyneoplastic changes. The amount of tissue, site of the sample, and orientation of the tissue on the slide are factors that may have contributed to the change in prevalence oftumors in these small endocrine organs. Further, in the earlier years, there may have been less emphasis on tumors of these small tissues compared to liver, lung, kidney, mammary gland, and other large tissues. The Good Laboratory Practices (GLP) regulations (23) may have improved the amount of tissue of the small organs available for histologic examination as indicated by the larger size of sections in studies (Table V). This may have facilitated histologic diagnoses of tumors in small organs and contributed to the increased prevalence of tumors. In B6C3Fl mice, where the common (> 10%) spontaneous tumors are primarily in large tissues such as liver, lung and lymphoid tissue, there were no major changes in the prevalence of tumors over a 9-yr period (14). Since several factors may influence the survival and tumor rates between laboratories and between studies within a laboratory, concurrent control group should be the primary basis for evaluation of carcinogenic potential of chemicals. However, relevant historical data of contemporary studies from the same laboratory may also be useful in such evaluations (8). In summary, an evaluation of 146 NCI-NTP 2-yr studies conducted over an ll-yr period revealed time-related decreases in survival and increases in body weight and prevalences of a number oftumor types in untreated F344/N rats. Decreasing trend in survival was probably due to an increase in body weight, increase in severity ofage-related nephropathy due to protein rich diet and changes in criteria for moribund sacrifice of aged animals. Increase in body weight appeared to be related to improvements in care, nutrition, and selection of animals and possibly subtle genetic changes in F344/N rats over the ll-yr period. At least 5 factors may have contributed to the increases in prevalence of tumors. These included: (a) changes in diagnostic criteria for leukemia, adrenal pheochromocytomas and pituitary tumors; (b) changes in the amount of tissue examined for thyroid, pituitary, and adrenal medulla tumors; (c) increase in body weight over time for mammary and pituitary tumors; (d) change in diet for thyroid tumors; and (e)interlaboratory variability. The time trends for the NCI-NTP historical database over an ll-yr period may not be unique

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