Beneficial Effects of NTP-2000 Diet on Growth, Survival, and Kidney and Heart Diseases of Fischer 344 Rats in Chronic Studies

Transcription

1 TOXICOLOGICAL SCIENCES 63, (2001) Copyright 2001 by the Society of Toxicology Beneficial Effects of NTP-2000 Diet on Growth, Survival, and Kidney and Heart Diseases of Fischer 344 Rats in Chronic Studies Ghanta N. Rao,*,1 Richard W. Morris, and John C. Seely *Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, 27709; Analytical Sciences, Inc., Durham, North Carolina 27713; and Experimental Pathology Laboratories, Research Triangle Park, North Carolina Received March 20, 2001; accepted July 17, 2001 Diet is one of the most important environmental factors influencing growth, survival, and appearance of age-associated diseases in rodents. NIH-07 open formula rodent diet was the selected diet for the National Toxicology Program studies from 1980 to After a number of experimental diets were evaluated, a new one designated as NTP-2000 was selected for rodents in NTP studies beginning in This report summarizes the results of dosed feed and inhalation studies for differences in growth, survival, and severity of kidney and heart lesions in Fischer 344 rats fed NTP-2000 or NIH-07 diets. In the dosed feed studies, male rats group housed and fed the NTP-2000 diet grew slightly slower, attained maximum body weight later, and lost less body weight by the end of the 2-year studies compared to the groups fed NIH-07. Female rats group housed and fed the NTP-2000 diet in dosed feed studies had significantly slower growth, with lower maximum body weight compared to the groups fed the NIH-07 diet. In the inhalation studies, male rats individually housed and fed the NTP-2000 diet had slightly higher maximum body weight and significantly higher final body weight, with lower loss of weight when compared to similarly housed groups fed the NIH-07 diet. In inhalation studies, female rats fed the NTP-2000 diet and individually housed had significantly slower growth. The NTP-2000 diet significantly increased the survival of male and female rats, with a dramatic increase in survival of males in inhalation studies. This diet also caused significant decreases in severity of nephropathy and cardiomyopathy, and the decrease was marked in males. These observations indicate that diets balanced for nutrients, such as the NTP-2000, could markedly improve the health and increase survival of the rats used in chronic studies. Key Words: diet; nephropathy; cardiomyopathy; body weight; survival; group housing; individual housing; Fischer 344 rat. During the last 20 years, the survival of most strains and stocks of rats commonly used for chronic carcinogenicity studies has been decreasing to less than 20% by the end of 2 years (Keenan et al., 1995). The number of animals at risk for carcinogenic effects of chemicals for 2 years decreases with decreasing survival and may decrease the sensitivity of the 1 To whom correspondence should be addressed at NIEHS, MD B3-08, P. O. Box 12233, Research Triangle Park, NC Fax: (919) rao@niehs.nih.gov. study. The decreasing survival is of concern for evaluation of safety and carcinogenic potential of pharmaceuticals, food additives, and industrial chemicals, because various regulatory agencies require 50% survival or 25 rats/sex at 24 months for negative studies (Keenan et al., 1995; Lang, 1991). The survival of Fischer 344 (F344/N) rats also decreased from more than 60% during the 1970s to less than 35% during the early 1990s, especially in male rats housed in chambers for inhalation studies (Haseman et al., 1985, 1998; Rao, 1995; Rao et al., 1990; Rao and Haseman, 1993). Causes considered to contribute to early mortality include faster growth and higher body weight leading to increased tumor incidences in both sexes and age-associated diseases such as nephropathy in males (Haseman et al., 1993). In F344 rats, leukemia is a lethal disease contributing to decreasing survival in 2-year studies (Haseman, et al., 1998). Diet is one of the most important environmental factors influencing aging diseases of rodents. Nephropathy in rats is listed under various disease conditions of the kidney, including protein-overload nephropathy and dietary nephritis (Montgomery et al., 1990), suggesting protein and diet are contributing to the severity of nephropathy. Diets containing 10 to 15% protein are considered to be adequate for growth and maintenance of rats (NRC, 1978, 1996). However, the most commonly used rodent diets such as NIH-07 contain 19 to 27% protein (Rao and Knapka, 1998). From 1980 to 1994, the NIH-07 open formula, nonpurified diet (Rao and Knapka, 1987) was the selected rodent diet for the National Toxicology Program studies to assess the toxic and carcinogenic potential of chemicals in rodents. This diet was formulated more than 25 years ago to enhance reproduction, growth, and lactation (Knapka et al., 1974). Some components of this diet, such as protein ( 24%), mineral content (Ca:P ratio), and vitamin concentrations (5000 IU of vitamin D/kg), may contribute to age-associated diseases. After a number of experimental diets with varying concentrations of protein, fat, fiber, minerals, and vitamins were evaluated (Rao et al., 1993, 1996), a new diet designated as the NTP-2000 open formula, nonpurified diet (Rao, 1996, 1997) was selected for all NTP rodent studies beginning in The purpose of this report is to summarize the differences in 245

2 246 RAO, MORRIS, AND SEELY growth, body weight, food consumption, survival, and kidney and heart lesions in F344 rats fed NTP-2000 or NIH-07 ad libitum. This report includes a summary of results from control groups of 2-year dietary (dosed feed) and inhalation studies sponsored by the NTP. MATERIALS AND METHODS Animals and animal care. Four-week-old F344 rats produced by defined microflora-associated (Charles River altered Shaedler microflora) breeders in a pathogen-free NTP production colony at Taconic Farms (Germantown, New York) were received at the facilities conducting the studies. After quarantine and acclimation for 2 weeks, they were determined to be pathogen-free, weighed, stratified by weight, and randomly distributed to groups. In dosed feed studies, rats of the same sex were housed 3 5 per cage in hanging-drawer type, polycarbonate cages with solid bottoms and sides (See-Through System II, Lab Products, Inc., Maywood, NJ). The cages were suspended from perforated stainless steel shelves covered with filter fabric. Heat-treated hardwood chips, used as contact bedding, were changed at 3 4 day intervals. In inhalation studies, the rats were housed individually in stainless steel, wire mesh cage batteries (Lab Products, Aberdeen, MD) suspended in 2-m 3 stainless steel chambers (Lab Products, Aberdeen, MD) throughout the study. Suspended wire mesh cages are essential for inhalation studies to assure uniform distribution of the test chemical throughout the inhalation chamber and uniform exposure of all animals in the chamber to the selected dose. This inhalationexposure system was described by Cheng and Moss (1989). Municipal drinking water was provided ad libitum by automated watering systems (Edstrom Industries, Milwaukee, WI). Animal use and care was in accordance with the United States Public Health Service policy on humane care and use of laboratory animals and the Guide for the Care and Use of Laboratory Animals (NRC, 1985, 1996). The studies were conducted in facilities accredited by the Association for Assurance and Accreditation of Laboratory Animal Care International, (Rockville, MD), with protocols approved by the institutional animal care and use committees of the facilities conducting the studies. Diets. The NIH-07 open formula, nonpurified diet (Rao and Knapka, 1987) in use for the NTP chemical toxicity and carcinogenicity studies since 1980 and the new NTP-2000 open formula, nonpurified diet (Rao, 1996, 1997) were the diets used for the studies included in this report. Ingredients and selected nutrient concentrations of the 2 diets are listed in Table 1. The NTP-2000 diet is different from the NIH-07 diet, with lower protein, higher fat, higher fiber, and lower Vitamin D concentrations, and with a higher calcium to phosphorous molar ratio. The rationale for selection of the ingredients and nutrient concentrations of the NTP-2000 diet was described in our previous reports (Rao 1996, 1997, in press). Each diet was in powdered (mash) form for the dosed feed studies or pelleted form for the inhalation studies (manufactured by Zeigler Bros., Inc., Gardners, PA) and was available ad libitum. In dosed feed studies, food consumption by cage was measured for 1 week at 4-week intervals for the 2-year period. Average food consumption (g/rat/day) for 2 years was computed for each dosed feed study. Each wire mesh cage battery, housing 16 to 24 rats in the inhalation studies, is equipped with a common wire mesh feed trough for all the rats of each cage battery. Due to considerable spillage/waste of feed from the common wire mesh feed trough, It was not possible to obtain useful food consumption data in the inhalation studies, and food consumption data were also not collected in the NTP inhalation studies. Two-year studies. Each 2-year study had one or more untreated or vehicle control groups and 3 or more treatment groups; however, only the data from untreated and vehicle control groups are included in this report. The chemical to be evaluated was administered by incorporation into the diet in the dosed feed studies, and by vapor, aerosol, or particles mixed in the breathing air of the inhalation studies. Start dates and diet for the dosed feed studies are listed in Table 2 and for inhalation studies in Table 3. Dosed feed studies were conducted in 3 different facilities located in the United States and all inhalation TABLE 1 Ingredients and Proximate Nutrient Composition of NTP-2000 and NIH-07 Open Formula, Nonpurified Diets Composition Amount by weight (%) NTP-2000 NIH-07 Nutrient Protein (%) a Fat (%) a Fiber (%) a Carbohydrates (%) b Energy (kcal/g) c Ingredient Ground corn Ground wheat Wheat middlings Soybean meal (49% protein) Fish meal (60% protein) Dried skim milk Corn gluten meal (60% protein) Alfalfa meal (17% protein) Oat hulls Purified cellulose Corn oil (without preservatives) Soy oil (without preservatives) Dried brewer s yeast Dry molasses Sodium chloride Calcium phosphate, dibasic (USP) Calcium carbonate (USP) Choline chloride (70% choline) d Methionine Vitamin premix 0.5 e f Mineral premix 0.5 g 0.25 h Note. Adapted from Rao, 1996 and a Mean of 76 lots of NTP-2000 diet and 77 lots of NIH-07 diet. b Other than crude fiber, calculated by difference including the correction for moisture and ash. c Physiological fuel value with no caloric value for fiber. d Included in the vitamin and mineral premix. e Vitamin premix per kg of NTP-2000 diet contained: vitamin A (stabilized palmitate or acetate), 4000 IU; vitamin D (activated animal sterol), 1000 IU; vitamin K (menadione sodium bisulfite complex), 1 mg; -tocopheryl acetate (vitamin E), 100 mg; folic acid, 1.1 mg; niacin, 23 mg; d-pantothenic acid (d-calcium pantothenate), 10 mg; riboflavin, 3.3 mg; thiamin (thiamin mononitrate), 4 mg; B12, 52 g; pyridoxine (pyridoxine hydrochloride), 6.3 mg; d-biotin, 0.2; with wheat middlings as carrier. f Included with the mineral premix. g Mineral premix per kg of NTP-2000 diet contained: magnesium (oxide), 514 mg; iron (sulfate), 35 mg; zinc (oxide), 12 mg; manganese (oxide), 10 mg; copper (sulfate), 2 mg; iodine (calcium iodate), 0.2 mg; chromium (acetate), 0.2 mg; with calcium carbonate as carrier. h Vitamin and mineral premix composition of NIH-07 diet was given by NRC (1978). studies were conducted in one facility. All of the testing facilities followed standardized NTP experimental procedures (NTP, 1992). Route of vehicle administration, special housing, and husbandry procedures associated with each route of administration may influence growth and survival (Haseman and Rao, 1992; Haseman et al., 1997, 1998). Therefore, more than 10 studies using

3 BENEFICIAL EFFECTS OF NTP-2000 DIET ON RATS 247 TABLE 2 Year and Month of Study Start with Maximum Body Weight, Survival, and Food Consumption for Dosed Feed Studies Year Month Maximum body weight (g) a Survival (%) b Food consumption (g/rat/day) a NIH-07 diet, males c d d d d Mean SE NTP-2000 diet, males d,e d d c,d Mean SE NIH-07 diet, females c d d d d Mean SE NTP-2000 diet, females d,e c c c,d Mean SE Note. 50/group unless stated otherwise. No NTP dosed-feed study started in a Mean SD. b Kaplan-Meier estimate. c 60/group. d Studies evaluated for severity of nephropathy and cardiomyopathy. e 100/group. the NIH-07 diet started during the period 1990 to 1994, and involving dosed water, gavage, and skin painting methods of chemical administration, were not included in this report. Furthermore, studies with the NTP-2000 diet started during the period 1994 to 1998, using dosed water, gavage, or a skin painting method of administration were too few in number by each route to provide reliable statistical comparison with the corresponding methods of administration using the NIH-07 diet. Growth and body weight. Rats were weighed at 4-week intervals throughout the study. Growth patterns were determined by computing averages for body weights (see Statistical procedures for details) by diet and route of administration. Group mean weights at week 1 and years 1.0 and 1.5, the maximum mean body weights attained during the course of the study, final body weights, and the maximum body weight gains were the measures chosen for assessing the difference in body weights between diets. Lesions. Nephropathy and cardiomyopathy are the most common nonneoplastic lesions in most strains and stocks of rats used for chronic toxicity and carcinogenicity studies. Evaluation of degree of severity of nephropathy and cardiomyopathy is not a routine procedure for the NTP studies. To assess the effect of diet on severity of these lesions, control groups from 4 studies for each diet were graded for the severity of nephropathy and cardiomyopathy. All the rats that survived for more than 21 months of the 2-year studies were included in this evaluation. For the NIH-07 diet, 146 male and 170 female rats, and for the NTP-2000 diet, 202 male and 208 female rats (one of the studies with the NTP-2000 diet included one control group of 100/sex) were evaluated. TABLE 3 Year and Month of Start with Maximum Body Weight and Survival for Inhalation Studies Year Month Maximum body weight (g) a Survival (%) b NIH-07 diet, males Mean SE NTP-2000 diet, males Mean SE NIH-07 diet, females Mean SE NTP-2000 diet, females Mean SE Note. 50/group; no NTP inhalation study started in 1995 or a Mean SD. b Kaplan-Meier estimate.

4 248 RAO, MORRIS, AND SEELY TABLE 4 Criteria for Grading the Severity of Nephropathy and Cardiomyopathy Severity Grade Description Nephropathy Minimal 1 Less than 10% of organ affected with presence of focal to multifocal regenerative renal tubules surrounded by a thickened basement membrane. Mild % of organ affected with multifocal renal tubule regeneration, glomerular thickening, tubular protein casts and chronic inflammatory cell infiltration. Moderate % of organ affected with more diffuse involvement with greater severity of the aforementioned changes. Variable amounts of interstitial fibrosis. Marked 4 80% 100% of organ affected with nearly entire renal parenchyma involved. Greater severity of interstitial fibrosis and tubular protein casts. Occasional mineralization of basement membranes. Cardiomyopathy Minimal 1 Less than 10% of organ affected focal to multifocal combination (any) of myofiber degeneration/necrosis, fibrosis, and mononuclear cell infiltration. Some interstitial branching of lesions. Lesions often noticed in apex, papillary muscles, and free wall of left ventricle. Mild % of organ affected. Greater severity of changes noted in grade 1. Discrete bundles of fibrosis and inflammation. Moderate % of organ affected with more extensive changes and may contain myofiber atrophy and occasional mineralization. Marked % of organ affected with possible pathological evidence of heart failure. Criteria for grading the severity of the lesions are listed in Table 4. A pathologist (J.C.S.) who had no prior association with the studies determined the severity of the lesions, and the lesions were evaluated without prior knowledge of the diets used for each study. The years of study start include 1 in 1991, 1 in 1992, and 2 in 1994 for the NIH-07 diet, and 1 in 1994, 2 in 1995, and 1 in 1996 for the NTP-2000 diet (Table 2). There were no NTP studies started in 1993 using the dosed feed route. Only 4 of the NTP studies using the NTP-2000 diet were complete with pathology at the time of this evaluation. Therefore, only these studies were compared with 4 studies of rats fed the NIH-07 diet for differences in severity of these lesions. The NIH-07 diet studies included in this evaluation represent studies done in the most recent past for appropriate comparison with the NTP-2000 diet groups. Inhalation studies were not included in this evaluation. Statistical procedures. The individual study was treated as the experimental unit for purposes of data summary and comparisons between diets. For growth data, food consumption, and survival the mean for each study is computed and measures of variability reflect variation among individual studies. We selected SE (standard error) over SD (standard deviation) because SE is an appropriate measure of variation associated with the means of several studies with each diet. The SD would be a more appropriate measure of variation for a single study or group (Steel and Torrie, 1980), and this measure of variance was included in Tables 2 and 3 for each study. Growth data. For each study, individual rat body weights were measured at 4-week intervals from the beginning of the study until the rat was removed from the study. Simple linear interpolation was used to compute body weight for each animal for the weeks in which body weights were not taken. This was done to ensure that body weight summaries at 1.0 and 1.5 years included data from all groups. Final body weights of control groups are from week 104, except in dosed feed studies where final weights of 2 groups on the NIH-07 diet and 1 group of 100 on the NTP-2000 diet were taken at 105 weeks and 1 group on the NIH-07 diet at 103 weeks. Means SE of body weights at week 1, and years 1.0, 1.5, and 2 were computed for each study. The maximum mean body weight, defined as the highest mean body weight attained by the group of control rats over the course of the study, was also computed, along with the week in which the maximum body weight was attained. Maximum body weight gains were computed by deducting the body weight at week 1 from the maximum mean body weight. Differences between diets in mean body weights at week 1, and 1.0, 1.5, and 2 years were tested using a 2-tailed t-test at Diet-related differences in maximum mean body weight, in the week maximum weight was attained and the maximum body weight gains, were also tested by a 2-tailed t-test (Steele and Torrie, 1980). These tests were carried out as preplanned comparisons, with no adjustment for multiple comparisons. Additional tests on body weight, which compared diets with respect to the difference between maximum mean and final mean weight, were also done using a 2-tailed t-test. These post hoc tests, however, were judged significant at a lower probability level, and in 2 cases where evidence of variance heterogeneity was present the degrees of freedom for the t statistic was adjusted downward using Satterwaite s approximation (Steele and Torrie, 1980). Survival data. Kaplan-Meier estimates of the survival function (Hosmer and Lemeshow, 1999) were used to summarize survival. For purposes of estimation, rats removed from the study by sacrifice at study termination, accidentally killed, or discovered missing over the course of a study were treated as censored deaths. Animals removed from the study because of natural death or moribund sacrifice were treated as uncensored deaths. Survival estimates were obtained at 103 to 105 weeks of study. The log-rank test (Hosmer and Lemeshow, 1999), which uses individual animal data, was employed to test for differences between diets. Computations were carried out using the Lifetest procedure (SAS/STAT, 1999). Time trends. Jonckheere s test (Hollander and Wolfe, 1999) was used to evaluate the chronological trend in body weight, food consumption, and survival among studies by diet, sex, and route of administration. Kidney and heart lesions. Cumulative multinomial probabilities reflecting ordered severity categories of 1 (minimal), 2 (mild), 3 (moderate), and 4 (marked) histopathology scores for nephropathy and cardiomyopathy were tested for the difference between diets, using a likelihood ratio test in the GENMOD procedure of SAS with a cumulative logit-link function. Food Consumption RESULTS Food consumption for male and female rats in dosed feed studies is included in Tables 2 and 5. Male rats fed the NTP-2000 diet consumed significantly (p 0.05) more feed than male groups fed the NIH-07 diet; the difference was 1.36

5 BENEFICIAL EFFECTS OF NTP-2000 DIET ON RATS 249 FIG. 1. Growth patterns of male and female Fischer 344 rats in dosed-feed and inhalation studies fed either NTP-2000 (broken line) or NIH-07 (solid line) diet for 2 years. g/day or 8.5% more for NTP-2000 diet groups. Female rats fed the NTP-2000 diet also consumed significantly (p 0.05) more feed than the groups fed the NIH-07 diet. However, the difference was only 0.72 g/day or 6.4%. Growth and Body Weights Dosed feed studies. Average growth curves for all the control groups of male and female rats of dosed feed studies for the NTP-2000 and NIH-07 diets are presented in Figure 1. The growth patterns for both diets were essentially the same for each sex, except that the body weight loss at the end of the study for males and the body weight gain for females during the last 3 months of the study were not as pronounced in the NTP-2000 diet groups. Group mean body weights at 1 week, 1.0 year, and 1.5 years, maximum mean body weight attained during the course of the studies, final body weights, and maximum body weight gains for both diets are listed in Tables 2 and 5. The body weights of groups of male rats fed the NTP-2000 diet, up to and including maximum body weight, were lower but not statistically different (p 0.05) from those of the groups fed the NIH-07 diet. However, the final body weights of the male rats fed the NTP-2000 diet were higher, but not significantly different (p 0.05), from those of the groups fed the NIH-07 diet (Table 5). Male rats fed the NTP-2000 diet attained maximum mean body weight at approximately 76 weeks of the study compared to 67 weeks for NIH-07 diet groups. The age for reaching maximum mean body weight was significantly greater and different (p 0.05) for the male rats fed the NTP-2000 diet compared to the NIH-07 diet groups, but the maximum weight gains were not different. The body weights of groups of female rats fed the NTP-2000 diet at 1 week, 1.0 year, and 1.5 years, the maximum mean body weights, as well as the group mean body weights at the end of the 2-year studies were significantly lower (p 0.05) than those of the groups fed the NIH-07 diet. The maximum body weight gain of groups fed the NTP-2000 diet was also lower but not significantly different (p 0.05) from that of the groups fed the NIH-07 diet. The groups fed the NTP-2000 diet attained maximum body weight at approximately 100 weeks of the study, compared to 97 weeks for the groups fed the NIH-07 diet. The ages for reaching maximum mean body weight were not significantly different (p 0.05) for female rats fed NIH-07 or NTP-2000 diets. Inhalation studies. Average growth curves for all the control groups of male and female rats in the inhalation studies fed NTP-2000 or NIH-07 diets are presented in Figure 1. The growth patterns of male and female rats for both diets were similar to the dosed feed studies. Mean body weights at 1 week, 1.0 year, and 1.5 years, maximum mean body weights attained during the course of the studies, final body weights, and maximum body weight gains for both diets of the inhalation studies are listed in Tables 3 and 5. In inhalation studies, the body weights of male rats at the 5 time points listed in Table 5 were higher than in the dosed feed studies. The body weights at 1.5 years and the maximum body weights of groups fed both diets were significantly (p 0.05) higher than the respective diet groups at the above points in the dosed feed studies (Table 5). The maximum body weight gain and the final body weight of the groups fed the NTP-2000 diet in inhalation studies were also significantly (p 0.01) higher than the respective body weights of groups fed the NTP-2000 diet in the dosed feed studies. The body weights of female rats in inhalation studies at all the selected time points except at week 1 were higher than those of the same diet groups at the corresponding time points in dosed feed studies for both the NIH-07 and NTP-2000 diet groups (Table 5). The body weights of groups of male rats fed the NTP-2000 diet at week 1 were significantly lower (p 0.01) than the groups fed the NIH-07 diet. The mean body weight at 1.5 years, and the maximum weight of male rats fed the NTP-2000

6 250 RAO, MORRIS, AND SEELY TABLE 5 Survival, Body Weights, and Food Consumption of Fischer 344 Rats Fed NIH-07 or NTP-2000 Diet for 2 Years Male Female Observation NIH-07 NTP-2000 NIH-07 NTP-2000 Dosed feed studies a Survival (%) ** ** 2.1 Body weights (g) At 1 week * * 2.1 At 1.0 year * 5.3 At 1.5 years ** 5.8 Maximum ** 5.6 Final ** 5.6 Weight gain b Food consumption (g/rat/day) * * 0.24 Inhalation studies c Survival (%) ** ** 1.6 Body weights (g) At 1 week ** ** 2.7 At 1.0 year ** 3.4 At 1.5 years * 4.9 Maximum Final * Weight gain b * a Mean standard error (SE) of 8 control groups of 8 studies with 405 rats for the NIH-07 diet and mean SE of 9 control groups of 7 studies with 495 rats for the NTP-2000 diet. b Maximum body weight gain (maximum body weight less week-1 body weight). c Mean SE of 5 control groups of 5 studies with 244 rats for the NIH-07 diet and mean SE of 6 control groups of 6 studies with 290 rats for the NTP-2000 diet. *p **p diet were slightly but not significantly (p 0.05) higher than the mean body weights of the groups fed the NIH-07 diet in the inhalation studies. However, the maximum weight gains and the final body weights of the male groups in inhalation studies fed NTP-2000 were significantly (p 0.05) higher than those of the groups fed the NIH-07 diet. Male rats fed the NTP-2000 diet attained maximum mean body weight at approximately 86 weeks into the study compared to 79 weeks for NIH-07 diet groups. The age for reaching maximum mean body weight was not significantly different (p 0.05) for male rats fed different diets in inhalation studies. The body weights of female rats in inhalation studies fed the NTP-2000 diet at 1 week, 1.0 year, and 1.5 years were significantly (p 0.05) lower than for the groups fed the NIH-07 diet. The maximum mean body weight as well as the final body weight of female rats fed the NTP-2000 diet were lower but not significantly different (p 0.05) from those of the groups fed the NIH-07 diet. The groups fed the NTP-2000 diet attained maximum body weight at approximately 99 weeks of the study compared to 97 weeks for the groups fed the NIH-07 diet. The age for reaching maximum mean body weight was not significantly different (p 0.05) for female rats of the inhalation studies fed NIH-07 or NTP-2000 diets (Table 3) and the diets did not affect the maximum body weight gain. Survival Probability of survival plots for all the control groups of male and female rats of the dosed feed studies fed NTP-2000 and NIH-07 diets are presented in Figure 2. In groups receiving the NTP-2000 diet, mortality of both males and females was delayed and the mortality rate was markedly lower than that of the groups fed the NIH-07 diet. Survivals for each study and average survival at 2 years for both diets are given in Tables 2 and 5. Survival of both male and female groups of rats fed NTP-2000 were markedly and significantly (p 0.01) higher than in the groups fed NIH-07. Survival plots for all the control groups of male and female rats of the inhalation studies for the NTP-2000 and NIH-07 diets are shown in Figure 3. Mortality was delayed and the mortality rate was markedly lower in groups fed the NTP-2000 diet. There was a substantial delay in mortality of male rats fed NTP-2000, leading to a marked increase in survival compared to those fed the NIH-07 diet. Survivals for each study and average survival at 2 years for both diets of the inhalation studies are listed in Tables 3 and 5. Survival of both male and female rats fed the NTP-2000 diet was markedly and significantly (p 0.01) higher than in the groups on the NIH-07 diet. In inhalation studies, the average survival of male groups fed

7 BENEFICIAL EFFECTS OF NTP-2000 DIET ON RATS % of the male rats showing a severity of less than 3 and with lesions in less than 40% of the kidney tissue. The severity of nephropathy in female rats fed the NTP-2000 diet also decreased, with only 12% of the females having lesions in more than 10% of the kidney tissue compared to lesions in 10% to 79% of the kidney tissue in 40% of the females fed the NIH-07 diet. The severity grades were for the NIH-07 diet and for the NTP-2000 diet groups. The severity was significantly (p 0.001) lower in the NTP-2000 diet groups. Cardiomyopathy Distribution of severity grades of cardiomyopathy for male and female rats fed NTP-2000 or NIH-07 diets are presented in Figure 5. In male rats fed the NTP-2000 diet, severity of cardiomyopathy decreased with almost all ( 98%) the male rats showing a severity of less than 3, and with lesions in less than 40% of the heart muscle. Male rats fed the NIH-07 diet were more frequently associated with grade 3 cardiomyopathy FIG. 2. Kaplan-Meier survival estimates for male and female Fischer 344 rats in dosed-feed studies fed either NTP-2000 (broken line) or NIH-07 (solid line) diet for 2 years. the NTP-2000 diet was almost twice that of the groups fed NIH-07. Time Trends There were no significant (p 0.05) chronological trends in body weight, feed consumption, or survival among studies by diet and sex in dosed feed studies or in inhalation studies. Nephropathy Patterns of nephropathy by severity grade for male and female rats fed NTP-2000 or NIH-07 diets are shown in Figure 4. The severity grades were (mean SE) for the NIH-07 diet and for the NTP-2000 diet groups. The severity was significantly (p 0.001) lower in the NTP diet groups. Approximately 53% of the male rats fed the NIH-07 diet had severity grades of greater than 2 with lesions in 40 to 80% of the kidney tissue. In male rats fed NTP-2000, the severity of nephropathy decreased, with approximately FIG. 3. Kaplan-Meier survival estimates for male and female Fischer 344 rats in inhalation studies fed either NTP-2000 (broken line) or NIH-07 (solid line) diet for 2 years.

8 252 RAO, MORRIS, AND SEELY FIG. 4. Distribution of severity of nephropathy grades for male and female Fischer 344 rats fed NTP-2000 or NIH-07 diet for 2 years. See Table 4 for description of severity grades. (with lesions in more than 40% of the heart muscle) in 13% of the male rats. Furthermore, the proportion of male rats in the NTP-2000 diet groups with minimal severity (lesions in less than 10% of the heart muscle) more than doubled (35 vs. 16%) compared to the male rats fed the NIH-07 diet. The severity grades were (mean SE) for the NIH-07 diet and for the NTP-2000 diet groups. The severity was significantly (p 0.001) lower in the NTP-2000 diet groups. The severity of cardiomyopathy in the female rats fed the NTP-2000 diet decreased also, with a decrease in the proportion of rats having severity grade 2 (lesions in 10 to 39% of the heart muscle) from 29% in NIH-07 diet groups to 12% in NTP-2000 diet groups. The severity grades were for the NIH-07 diet and for the NTP-2000 diet groups. The severity was significantly (p 0.001) lower in the NTP-2000 diet groups. DISCUSSION The NTP-2000 diet contains 14% protein with a balanced amino acid pattern (Rao, 1996, 1997). The fiber content of the NTP-2000 diet is higher (Table 1) than in diets formulated for FIG. 5. Distribution of severity of cardiomyopathy grades for male (left panels) and female Fischer 344 rats fed NTP-2000 or NIH-07 diet for 2 years. See Table 4 for description of severity grades.

9 BENEFICIAL EFFECTS OF NTP-2000 DIET ON RATS 253 maximum growth (Rao and Knapka, 1998). The additional fiber is from cellulose and oat hulls, which is insoluble and not fermented by bacteria, therefore not expected to contribute to calories or loss of nitrogen (NRC, 1995). Fiber from cellulose and oat hulls, up to 20% in the diet, do not affect growth, because the feed intake will increase to compensate for dilution of the diet by fiber (NRC, 1995). The rats in dosed feed studies fed the NTP-2000 diet consumed more food, possibly to compensate for higher fiber content of the NTP-2000 diet (Rao, 1996). The body weights of groups fed NTP-2000 were significantly (p 0.05) lower than those of the groups fed NIH-07 at week 1, irrespective of sex and housing conditions. According to the standardized procedures for the NTP studies (NTP, 1992), the diet fed to rats during quarantine and acclimation from 4 to 6 weeks of age shall be the same as the diet fed during the 2-year study. Due to lower protein content, the NTP-2000 diet appears to be preventing fast growth. Therefore, week 1 body weights of rats fed NTP-2000 were lower than those fed NIH-07. Reeves et al. (1979) reported that growth of weanling rats fed a diet with 10% protein and 10% fat was slower but not markedly different from growth of the rats fed 20% protein and 10% fat diet for a 30-day period. Our observations with NIH-07 and NTP-2000 diets are in agreement with the above report (Reeves et al., 1979), indicating that 14% protein concentration in the NTP-2000 diet is closer to optimum for growth and 20% or higher protein concentration is not necessary for growth and maintenance. The male rats in dosed feed studies fed the NIH-07 diet appear to have grown faster, attained maximum body weight earlier, and lost more body weight by the end of the 2-year studies when compared to the groups fed the NTP-2000 diet. The NTP-2000 diet slowed the growth and lowered the maximum body weight of female rats, but the decrease in maximum body weight was less than 7%. Even though the body weights of male rats in inhalation studies, individually housed and fed the NTP-2000 diet, were significantly lower at week 1. The body weight gain of these groups of male rats was significantly higher than those of the groups fed the NIH-07 diet. However, higher maximum body weights due to the NTP-2000 diet did not lead to loss of more body weight at 2 years compared to the NIH-07 diet groups. On the contrary, the loss of body weight in the NTP-2000 diet groups at 2 years was 4%, compared to more than 9% in the NIH 07 diet groups. The female rats individually housed in inhalation studies and fed the NTP-2000 diet had slightly slower growth up to 1.5 years, but the maximum and final body weights and the maximum body weight gains were close to those of the groups fed the NIH-07 diet. In general, both male and female rats fed either diet and individually housed in inhalation studies grew faster and attained significantly higher maximum body weights than rat groups in the dosed feed studies. The females fed the NIH-07 diet, whether group housed or individually housed grew faster and attained higher maximum body weight than the females fed the NTP-2000 diet. However, compared to similarly housed males fed NTP-2000 diet, males fed the NIH-07 diet had faster growth and higher maximum body weight only in the group housed, dosed feed studies. The final body weights of males fed the NIH-07 diet and housed individually or in groups were significantly different, and approximately 9% lower than the maximum body weight. However, the final body weight of the groups fed the NTP diet and caged either individually or in groups was not significantly different and only slightly lower than the maximum mean body weight in the corresponding caging system. These observations indicate that the NIH-07 diet causes significant loss of body weight in the male rats, whether they are group housed or individually caged. The NTP-2000 diet increased the weight gain of individually housed males and not the group housed males; and decreased the weight gain of group housed females and not the individually housed females, compared to NIH-07 diet groups housed in corresponding caging systems. These observations indicate that there were interactions between diet, caging system, and sex affecting the growth patterns and body weights of study rats. Survival at 2 years was markedly higher in males and females fed the NTP-2000 diet compared to NIH-07 diet groups, irrespective of individual or group housing and maximum body weight. The increased survival due to the NTP diet was more marked in males. The survival of individually housed males fed NTP-2000 was almost twice that in the groups fed NIH-07, even with similar or slightly higher maximum body weights. The survival of male as well as female rats individually housed was significantly lower than for group housed rats fed the same diet. This observation was in agreement with our previous report (Rao, 1995). The reason for lower survival of individually housed rats is not known. It was hypothesized that individual housing may cause stress leading to increased serum corticosteroids. Prolonged stress depletes the Leydig cell (testicular interstitial cell) enzyme that inactivates glucocorticoids leading to impaired steroidogenesis and pituitary cell proliferation, including neoplasms in male rats (Nyska et al., 1998). However, diet or caloric restriction was reported to cause hyperadrenocorticism (Klebanov et al., 1995), but it will not lead to increased neoplasms and lower survival at 2 years. Since individually housed rats had higher maximum body weight and lower survival than group housed rats, higher body weight may be considered as a contributing factor for higher mortality. However, the survival of individually housed male rats with higher body weight fed the NTP diet was significantly higher than the group housed male rats fed NIH-07. These results indicate that individual or group housing appears to be interacting with diet to influence survival. The greatest increase in survival was observed in NTP fed male rats with the highest maximum body weight, indicating that the higher weight may not be the major contributing factor for mortality. Because the NIH-07 diet was replaced by the NTP-2000 diet

10 254 RAO, MORRIS, AND SEELY in 1994, diet comparisons of growth and survival data may reflect differences in the chronological time at which studies were done (see Tables 2 and 3). This is the reason we have selected approximately an equal number of the NIH-07 control groups from the most recent past to compare with the NTP groups. In dosed feed studies, the observed decreases in body weight and increases in survival of rats fed NTP-2000 contrast with the NTP experience of increasing body weights and decreasing survival of rats fed NIH-07 from 1980 to 1994 (Haseman et al., 1985, 1998; Rao et al., 1990). Although change of diet did not substantially change the growth patterns and body weights in inhalation studies, the NTP-2000 diet caused more marked increase in survival in inhalation studies than in the dosed feed studies. These observations suggest that any underlying time-related trends, especially on survival, have been altered by the change in diet. Modifying the diet to decrease protein consumption could decrease the severity of nephropathy (Rao et al., 1993, 1996). The NTP-2000 diet, with adequate concentration of protein for growth and maintenance, markedly decreased the severity of nephropathy and improved the health of the kidney compared to the NIH-07 diet with excess protein. Improving the health of the kidney possibly prevented lesions secondary to nephropathy (Montgomery et al., 1990) including cardiomyopathy and decreased the mortality of both male and female rats in 2-year studies. In addition, the NTP-2000 diet contains approximately 3% more fat from corn oil. High corn oil intake either by gavage or through diet appears to decrease the incidence or delay the development of leukemia, a lethal tumor in F344 rats (Hursting et al., 1994; NTP, 1994; Rao, 1994, 1996; Rao et al., 1996; Rao and Haseman, 1993). Increased fat consumption due to corn oil in the NTP-2000 diet will comprise approximately 1.3 ml/kg of the body weight of a rat. This is lower than the 2.5 ml of corn oil or safflower oil/kg body weight causing a decrease in the incidence of leukemia (NTP, 1994). The contribution of corn oil at 1.3 ml/kg body weight to decreasing leukemia and thus increasing survival is not known at this time. Evaluation of differences in tumor incidences of control groups of rats fed the NTP-2000 diet and the NIH-07 diet is in progress. Diets balanced for nutrients such as the NTP-2000 diet could markedly improve the health of rats in 2-year studies and substantially decrease mortality so that the objectives of chronic carcinogenicity studies can be accomplished. Diet or caloric restriction could also decrease the severity of nephropathy and cardiomyopathy with marked increase in survival of rats in chronic studies (Iwasaki et al., 1988; Maeda et al., 1985). However, diet or caloric restriction is an involuntary process and can lead to hyperadrenocorticism (Klebanov et al., 1995). Diet restriction is not practical and may not be appropriate for dosed feed studies. If a test chemical influences the energy utilization of animals by either biochemical or behavioral changes (such as stimulants, sedatives, anabolic agents, antibiotics), diet, or caloric restriction may disproportionately change the physiological processes in treatment groups, complicating the interpretation of chemical effects. Diet or caloric restriction of more than 10% may require individual housing, but the results of 2-year studies included in this report show that group housing increases survival, suggesting that group housing promotes the overall well-being of rats. Modification of diet with balanced nutrients alone will accomplish the same major beneficial effects of diet or caloric restriction so that the objectives of chronic toxicity and carcinogenicity studies can be accomplished. ACKNOWLEDGMENTS The authors thank Dr. Charles Alden for editing the manuscript and Drs. Ronald Herbert and Gordon Flake for scientific review of the manuscript. REFERENCES Cheng, Y. S., and Moss, O. R. (1989). Inhalation exposure systems. In Concepts in Inhalation Toxicology (R. O. McClellan and R. F. Henderson, Eds.), pp Hemisphere Publishing, New York. Haseman, J. K., Boorman, G. A., and Huff, J. (1997). Value of historical control data and other issues related to the evaluation of long-term rodent carcinogenicity studies. Toxicol. Pathol. 25, Haseman, J. K., Eustis, S. L., and Ward, J. M. (1993). Contributing causes of death and utilization of this information in the statistical evaluation of tumor data. 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