Composition of diet may influence growth, diseases, tumor rates, life span and responses to chemical treatment. Contributing causes for mortality of rats in long-term studies that may be influenced by diet include nephropathy in males, mammary tumors in females and anterior pituitary tumors in both sexes of most strains or stocks of rats and leukemia in Fischer 344 (F344) rats (Haseman et al. 1993). From 1980 to 1994, the NIH-07 open-formula nonpurified diet (Rao and Knapka 1987) with high protein (~24%), low fat (~5%) and low fiber (~3.5%) was the selected diet for the National Toxicology Program (NTP)-sponsored chemical toxicity and carcinogenicity studies in rodents. The NIH-07 diet was formulated more than 20 y ago for reproduction, lactation and growth of rodents (Knapka et al. 1974). Some aspects of this diet, such as a low calcium:phosphorus (Ca:P) ratio and high protein content, may have contributed to nephrocalcinosis, severity of nephropathy and other lesions. Since 1988, a number of diets have been formulated and evaluated to understand the influence of dietary protein, fat and fiber on chronic diseases and tumor incidences. Based on the results of the experimental diets, an open-formula nonpurified diet designated as NTP-2000 was formulated. The purpose of this report is to provide a summary of the results with various experimental diets and describe the formulation and composition of the NTP-2000 diet. This report also includes a summary of results of a 13-wk study with the NTP-2000 diet.

EXPERIMENTAL DIETS

Growth patterns and maximum body weights attained by each sex fed the 23% (NIH-07) or 15% (NTP-88) protein diets were not significantly different (Rao et al. 1993). Decreasing the protein content of diet from 23% to 15% decreased protein consumption by 30%. Severity of nephropathy in male rats was significantly higher when the rats were fed a 23% protein diet (moderate to marked) compared with the 15% protein diet (minimal to mild). Severity of nephropathy in female rats also decreased with decreased protein consumption. The 24-h urine volume, proteinurea and water consumptions were significantly lower in groups fed 15% protein diet. This study indicated that a nonpurified diet containing 15% protein from mixed animal and cereal grain sources supplemented with methionine is adequate for growth and maintenance. Decreasing the protein content of diet decreased the severity of nephropathy without decreasing caloric intake or body weight gain.

Corn oil, when given as gavage at 5 mL/kg body wt for 2 y, increased body weight gain and pancreatic acinar cell tumors of male F344 rats. However, corn oil gavage decreased leukemia and leukemia-associated mortality, which resulted in higher survival of male rats (Rao and Haseman 1993). Corn oil and safflower oil, when given as gavage at 2.5 to 10 mL/kg body wt to male F344 rats for 2 y, caused a dose-related decrease in the incidence of leukemia (NTP 1994). These results indicated that higher intake of fat (as corn oil or safflower oil) than the fat available from the NIH-07 diet may be beneficial to F344 rats.

A nonpurified experimental diet with lower (~15%) protein, higher (~7.2%) fat and higher (~9.4%) crude fiber (NTP-90 diet) than the NIH-07 diet with approximately the same physiological fuel value (assuming no energy value for fiber) as the NIH-07 diet was formulated and evaluated (Rao et al. 1996). Crude fiber content of the NTP-90 diet was increased to compensate for higher energy due to higher concentration of fat. Growth patterns were similar for rats fed the two diets except the adult body weights of groups fed the NTP-90 diet were ~6% lower than those of rats fed the NIH-07 diet. There were no differences in hematology parameters of the rats fed the two diets. Except for blood urea nitrogen (BUN), serum triglyceride and serum alkaline phosphatase (ALP) values, there were no differences in clinical chemistry parameters between the groups fed the two diets. Blood urea nitrogen values of NTP-90 diet-fed groups were lower because of lower protein consumption. Kidney weights and severity of nephropathy of groups fed the lower protein diet were significantly (P < 0.01) lower. Higher fat and/or fiber consumption decreased the incidences of leukemia, adrenal pheochromocytoma and adrenal medullary hyperplasia of male rats fed the NTP-90 diet. However, mesothelioma and mesothelioma-associated mortality increased in the male rats fed the NTP-90 diet (Rao et al. 1996). The incidence of mesothelioma was slightly higher than the maximum incidence observed in NTP studies using the NIH-07 diet, but most male rats with mesothelioma in the group fed the NTP-90 diet died before the end of the study. Mammary tumor-associated mortality decreased in females fed the NTP-90 diet. The NTP-90 diet seems to be an appropriate nonpurified diet for rats in long-term studies, but an increase in mesothelioma-associated mortality of male rats fed this diet was of concern.

To confirm the above observations with the higher fat and fiber diet (compared with the NIH-07 diet), two more nonpurified experimental diets designated as NTP-91 and NTP-92 with higher fat (~8.5%) and higher fiber (~14%) than the NTP-90 and NIH-07 diets were formulated and fed to groups of 60 rats of each sex for 2 y. The compositions of the NTP-91 and NTP-92 diets are essentially the same, except corn oil in the NTP-91 diet and safflower oil in the NTP-92 diet were the major sources of fat. An experimental diet with safflower oil was included because NTP evaluated safflower oil as a gavage vehicle in a 2-y study (NTP 1994). Growth patterns for rats fed the three diets were similar except the adult body weights of rats fed the higher fat and fiber diets (NTP-91 and NTP-92) were 6-8% lower with higher survival than in rats fed the NIH-07 diet (Rao et al. 1996). Kidney weights and severity of nephropathy were significantly lower in the groups fed the NTP-91 and NTP-92 diets. Serum concentrations of ALP and triglycerides and incidences of proliferative lesions of the adrenal gland were lower in the groups fed the experimental diets and are in agreement with the effects of the NTP-90 diet. However, higher fat and fiber diets (NTP-91 and NTP-92) did not cause an increase in mesothelioma and associated mortality, indicating that the higher mesothelioma incidence in the NTP-90 diet-fed male rats may be due to chance. The NTP-91 and NTP-92 diets delayed development of mammary tumors in females (Rao 1995, Rao et al. 1996) and confirmed the effect observed with the NTP-90 diet.

Results of studies with the experimental diets indicated that a 15% protein nonpurified diet is adequate for growth and maintenance of rats and that decreasing protein markedly decreased the severity of nephropathy. Increasing fat intake with fat similar to corn oil seems to decrease the incidence or severity of leukemia. Higher energy density of the higher fat diet, when adjusted with crude fiber to be closer to the energy density of the NIH-07 diet, decreased body weight gain. Increasing the fiber content of diet delayed mammary tumor development and associated mortality. Experimental diets with higher fat and fiber and lower protein (compared with the NIH-07 diet) decreased the incidences of adrenal medullary hyperplasia and adrenal pheochromocytomas in males and decreased the spontaneous tumor burden of rats in 2-y studies. These observations indicate that the diets for rats in long-term studies could be modified to decrease or delay chronic diseases and spontaneous tumor development.

A workshop was held at the National Institute of Environmental Health Sciences (Rao 1994) to discuss the results of the above studies, literature data and ingredients for formulation of a new diet to decrease the severity of chronic diseases and to delay development of spontaneous tumors in 2-y studies. A new diet designated as NTP-2000 has evolved from these efforts.

NEW DIET (NTP-2000)

Table 1. Ingredients and composition of NTP-2000 and NIH-07 open formula nonpurified diets1 [View Table]

Table 2. Nutrient concentrations of NIH-07 and NTP-2000 nonpurified diets1 [View Table]

Table 3. Selected observations of F344 rats fed NIH-07 or NTP-2000 nonpurified diet for 13 wk1 [View Table]

Vitamin D concentration was decreased from ~5000 to 1000 IU/kg, vitamin E level was higher due to higher fat content, vitamin K was changed from menadione to menadione sodium bisulfite complex and decreased, and thiamin and niacin levels were decreased, with no substantial changes in concentrations of other vitamins in the NTP-2000 diet compared with the NIH-07 diet. The Ca:P ratio along with other minerals and ingredients may contribute to mineralization at the corticomedullary junction of kidney (nephrocalcinosis) in female rats. In accordance with the AIN recommendation (Reeves et al. 1993), the Ca and P concentrations of the NTP-2000 diet were adjusted to have a Ca:P molar ratio of greater than 1) (to ~1.3). Sodium, potassium, iron, manganese and copper concentrations were decreased in the NTP-2000 diet compared with the NIH-07 diet. The nonpurified ingredients contribute ultratrace elements considered to be beneficial by AIN (Reeves et al. 1993) and NRC (1995). The NTP-2000 diet contained more than adequate concentrations of ultratrace elements (Rao 1996).

Body weights and food and water consumptions are given in Table 3. Growth patterns were essentially the same for both diet groups, except male rats fed the NTP-2000 diet had slightly slower growth rate for the first 6 wk (Rao 1996); however, the final (wk 14) body weights for both groups were the same (Table 3). Consumption levels for the NTP-2000 diet were 5-10% higher (P < 0.01) than those for the NIH-07 diet, and higher food consumptions may be due to higher fiber content of this diet. Water consumption of male rats fed NTP-2000 diet was lower (P < 0.05) than for the group fed the NIH-07 diet, and this difference may be related to decreased severity of nephropathy. There were no differences in hematological parameters (Rao 1996). There were no substantial differences in serum protein, creatinine, creatine kinase, bile acids, glucose, alanine aminotransferase or sorbitol dehydrogenase concentrations of male and female groups fed the NTP-2000 and NIH-07 diets (Rao 1996). There were some differences in BUN, triglycerides, cholesterol, ALP and 5-nucleotidase concentrations, and these values are listed in Table 3. Blood urea nitrogen levels of rats fed the NTP-2000 diet were lower due to lower protein consumption. Serum cholesterol concentration of male rats fed the NTP-2000 diet was higher, and ALP and 5-nucleotidase concentrations in male and female rats fed the NTP-2000 diet were lower than for the NIH-07 diet-fed groups, probably because of higher fat content of the NTP-2000 diet (Kim et al. 1976, Young et al. 1981). Even though the fat consumption of rats fed the NTP-2000 diet were higher than that of the rats fed the NIH-07 diet, the serum triglyceride concentrations of groups fed the NTP-2000 diet were lower, possibly due to the higher fiber content of this diet and possible hormonal (insulin, glucogan) changes associated with consumption of the NTP-2000 diet. There were no differences in serum Na, K or Ca concentrations in rats fed the two diets (Rao 1996). The serum phosphorus (P) concentration of male rats fed the NTP-2000 diet was slightly lower than that of the group fed the NIH-07 diet (71 vs. 81 ppm, P < 0.05), and this difference may be related to the lower P content of the NTP-2000 diet (Table 2).

There were no consistent and significant differences in the weights of heart, thyroid, pituitary, gonads or brain of rats fed the two diets. The liver, kidney and adrenal weights were significantly lower (P < 0.01) in rats fed the NTP-2000 diet (Table 3). Lower liver weight may be related to lower protein consumption and associated changes in induction of drug-metabolizing enzymes. Lower kidney weight may be related to decreased protein consumption-associated delay in nephropathy. Lower adrenal weights may be related to delay in early changes of medullary hyperplasia leading to pheochromocytoma. The most common lesions of F344 rats fed the NIH-07 diet for 13 wk were nephrocalcinosis in female rats, initial changes of nephropathy in male rats and early changes of cardiomyopathy in both sexes (Dixon et al. 1995). Incidences and severity of these lesions in the 13-wk study with the NTP-2000 and NIH-07 diets are given in Table 3. Nephrocalcinosis was observed in all female rats fed the NIH-07 diet, and the NTP-2000 diet prevented nephrocalcinosis. The NTP-2000 diet decreased the incidence and severity of nephropathy and decreased the severity of cardiomyopathy.

In conclusion, on the basis of 2-y studies with experimental diets containing lower protein and higher fat and fiber than the NIH-07 diet (Rao et al. 1996) and the 13-wk study with the NTP-2000 diet (Rao 1996), the NTP-2000 diet is adequate for growth and maintenance of rats. The NTP-2000 diet prevented nephrocalcinosis and seems to decrease the incidences or severity of diet- and possibly age-associated lesions.

ACKNOWLEDGMENTS