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New Diet (NTP-2000) for Rats in the National Toxicology Program Toxicity and Carcinogenicity Studies
FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.
32, 102–108 (1996)
0112
New Diet (NTP-2000) for Rats in the National Toxicology Program Toxicity and Carcinogenicity Studies GHANTA N. RAO National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received December 12, 1995; accepted March 30, 1996
New diet (NTP-2000) for Rats in the National Toxicology Program Toxicity and Carcinogenicity Studies. RAO, G. N. (1996). Fundam. Appl. Toxicol. 32, 102–108. Composition of diet may influence growth, diseases, tumor rates, and responses to chemical treatment. Since 1980 the NIH-07 open formula nonpurified diet has been the selected diet for the National Toxicology Program (NTP) toxicity and carcinogenicity studies in rodents. Studies with nonpurified experimental diets with lower protein and higher fat and fiber than the NIH-07 diet indicated that the diet for Fischer-344 (F344) rats in long-term studies could be modified to decrease the severity of chronic diseases and to decrease/delay the development of spontaneous tumors. Based on the results of these studies a new open formula nonpurified diet designated as NTP-2000 was formulated to contain Ç14.5% protein, Ç8.5% fat, and Ç9.5% fiber. Corn, wheat, and wheat middlings contribute to about 60% of the ingredients; soybean meal, fish meal, and alfalfa meal are the additional sources of protein; purified cellulose, oat hulls, and alfalfa meal are the major sources of fiber; and soy oil and corn oil are the major sources of fat in the NTP-2000 diet. The Ca:P ratio and mineral and vitamin concentrations were reformulated based on AIN-93 and NRC-95 recommendations. The NIH-07 and the NTP-2000 diets were fed to groups of 6-week-old F344 rats for 13 weeks and evaluated for growth patterns, food and water consumptions, hematology and clinical chemistry parameters, and organ weights and pathological changes. Growth patterns and body weights were similar for both diets. Food consumptions were slightly higher and water consumptions were slightly lower for the groups fed NTP-2000 diet. There were no differences in hematological parameters between the groups fed the above diets. Serum levels of cholesterol, alkaline phosphatase, and 5* nucleotidase were slightly higher in groups fed the NTP-2000 diet possibly due to higher fat content of this diet. However, the serum triglyceride levels were slightly lower in groups fed the NTP-2000 diet and it may be related to higher fiber content of the NTP-2000 diet. The liver and kidney weights of the groups fed NTP-2000 diet were significantly lower possibly due to lower protein content of this diet and lower protein consumption associated changes in Phase I and Phase II drug metabolizing enzyme systems. The adrenal weights were also lower in groups fed the new diet. The NTP-2000 diet prevented nephrocalcinosis and decreased the severity of nephropathy and cardiomyopathy, the common lesions of F344 rats in 13-week studies. These results indicate that the NTP-2000 diet is adequate for growth and main-
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Diet is an important factor influencing biological processes such as growth, survival, and diseases including tumor rates. Since 1980 the NIH-07 open formula nonpurified diet (Rao and Knapka, 1987) with high protein (Ç24%), low fat (Ç5%), and low fiber (Ç3.5%) has been the selected diet for the National Toxicology Program (NTP) sponsored chemical toxicity and carcinogenicity studies in rodents. This diet was formulated more than 20 years ago for reproduction, lactation, and growth of rodents (Knapka et al., 1974). Some components of NIH-07 diet such as high protein content may have increased the severity of nephropathy. Since 1988 a number of studies were conducted to obtain data on the influence of dietary protein, fat, and fiber (Rao et al., 1993; Rao and Haseman, 1993; NTP, 1994; Rao, 1994, 1995; Rao et al., 1996) on chronic diseases and tumor incidences. Decrease in protein consumption due to lower protein content of diet decreased the severity of nephropathy in rats without a significant effect on growth (Rao et al., 1993). In 2-year chemical carcinogenicity studies when corn oil was given by gavage (as a vehicle), there was a decrease in the incidences of leukemia, a lethal tumor in Fischer-344 (F344) rats. However, corn oil gavage significantly increased the adult body weight and pancreatic acinar cell tumors in male rats (Rao and Haseman, 1993; NTP, 1994). Higher fiber content of experimental diets appears to have delayed the development of mammary tumors in females (Rao, 1994, 1995; Rao et al., 1996). Higher fat or fiber of experimental diets decreased the incidences of adrenal medullary hyperplasia and adrenal pheochromocytomas in males (Rao, 1994, 1995; Rao et al., 1996). Lower protein and higher fat and fiber of experimental diets decreased the spontaneous tumor burden in 2-year studies (Rao et al., 1996). Results of these studies indicated that change in diet composition appears to be appropriate for rats in toxicity and carcinogenicity studies. A workshop was held at the National Institute of Environmental Health Sciences (NIEHS) in June 1993 to discuss possible changes in diet formulation and composition to de-
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TABLE 1 Ingredient Composition of NTP-20000 and NIH-07 Open Formula Nonpurified Diets Amount by weight (%) Ingredient
NTP-2000
NIH-07a
Ground wheat Ground corn Wheat middlings Soybean meal (49% protein) Fish meal (60% protein) Dried skim milk Corn gluten meal (60% protein) Dried brewer’s yeast Dry molasses Alfalfa meal (17% protein) Oat hulls Purified cellulose Corn oil (without preservatives) Soy oil (without preservatives) Sodium chloride Calcium phosphate, dibasic (USP) Calcium carbonate (USP) Choline chloride (70% choline) Methionine Vitamin premixc Mineral premixd
22.26 22.18 15.0 5.0 4.0 0.0 0.0 1.0 0.0 7.5 8.5 5.5 3.0 3.0 0.3 0.4 0.9 0.26 0.2 0.5 0.5
23.0 24.5 10.0 12.0 10.0 5.0 3.0 2.0 1.5 4.0 0.0 0.0 0.0 2.5 0.5 1.25 0.5 0.09b 0.0 0.125 0.125
nitrosamines, and chlorinated hydrocarbons (Rao, 1991). To decrease the contaminant levels of the new diet, limited amounts of these ingredients were used as protein sources. Additional protein from alfalfa meal and dried brewer’s yeast helped to complete the protein composition. Dried skim milk has casein as its major protein and casein may increase the severity of nephropathy in rats (Klahr et al., 1983). Furthermore, the availability of dried skim milk as an ingredient for laboratory animal diets is limited, so skim milk was not included in formulation of the new diet. Alfalfa meal is a good source of protein and fiber; oat hulls and beet pulp are good sources of fiber in experimental animal diets. Beet pulp was excluded since it has a high water-holding capacity and may disproportionately increase the bulk of intestinal contents (Rao, 1994). Alfalfa meal is a potential source of nitrosamines and oat hulls reduce pellet quality. High amounts of these ingredients may add high levels of phytates which may interfere with mineral absorption (FASEB, 1987). Thus, the proportion of either ingredient was less than 10% in the NTP-2000 diet. Approximately 50% of the crude fiber was from the above ingredients. Purified cellulose with the same specifications as the AIN93 diet (Reeves et al., 1993) was added to bring the crude fiber level up to the planned amount. The cereal ingredients and additional protein sources con-
a
Vitamin and mineral premix composition was given by AIN (1977). Included in the vitamin premix. c Wheat middlings as carrier. d Calcium carbonate as carrier. b
crease the severity of chronic diseases and to delay the development of spontaneous tumors in 2-year studies (Rao, 1994). A new diet designated as NTP-2000 has evolved from the above-mentioned efforts. This report provides (a) ingredient and nutrient composition of the new diet with explanations for their selection and (b) results of a 13-week study in F344 rats with the new diet. DIET COMPOSITION
Ingredients The ingredient composition of the NTP-2000 diet in comparison with the NIH-07 diet is provided in Table 1. Details of vitamin and mineral additions to NTP-2000 diet are listed in Table 2. This is a cereal-based nonpurified diet (American Institute of Nutrition, 1977) with corn, wheat, and wheat middling contributing to about 60% of the ingredients. Protein sources such as soybean meal, fish meal, and dried skim milk are commonly used in laboratory animal diets. Soybean products contribute to estrogenic activity due to naturally occurring phytoestrogens (Murphy, 1982). Fish meal can be a major source of contaminants such as heavy metals,
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TABLE 2 Vitamins and Minerals Added to the NTP-2000 Open Formula Nonpurified Dieta
Vitamins A D K a-Tocopheryl acetate Folic acid Niacin d-pantothenic acid Riboflavin Thiamin B12 Pyridoxine Biotin Minerals Magnesium Iron Zinc Manganese Copper Iodine Chromium a b
Amount
Source
4000 IU
Stabilized vitamin A Palmitate or acetate D activated animal sterol Menadione (MSBC)b
1000 IU 1 100 1.1 2.3 10 3.3 4 52 mg 6.3 0.2 514 35 12 10 2 0.2 0.2
d-Calcium pantothenate Thiamine mononitrate Pyridoxine hydrochloride d-biotin Magnesium oxide Iron sulfate Zinc oxide Manganese oxide Copper sulfate Calcium iodate Chromium acetate
mg/kg of finished diet. MSBC Menadione sodium bisulfite complex.
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TABLE 3 Proximate and Other Selected Nutrient Composition of NTP-2000 and NIH-07 Open Formula Nonpurified Diets % By weighta Nutrient
NTP-2000
NIH-07
Protein Fat Fiber Calcium Phosphorus Vitamin A (IU/kg) Thiamin (ppm) Carbohydrateb Energy (kcal/g)c Energy (kcal/g)d
14.6 { 0.91 8.2 { 0.5 9.3 { 0.81 0.94 { 0.06 0.58 { 0.04 6800 { 1500 12.1 { 1.45 52 3.77 3.40
22.9 { 0.44 5.4 { 0.26 3.4 { 0.39 1.18 { 0.07 0.94 { 0.04 6300 { 600 20.7 { 3.2 51 3.58 3.44
a
Mean { standard deviation of 15 contemporary lots. Other than crude fiber, calculated by difference including the correction for moisture and ash. c Physiological fuel value. d Physiological fuel value with no caloric value for fiber. b
tribute fat to this diet. Soy oil, corn oil, and stabilized animal fats are the common sources of added fat in laboratory animal diets. Corn oil was the most common fat source in the previous NTP studies (Rao and Haseman, 1993; NTP, 1994; Rao, 1994, 1995; Rao et al., 1996) and was found to decrease or delay the development of leukemia in male F344 rats. Soy oil was recommended by the American Institute of Nutrition (AIN), (Reeves et al., 1993) as the most appropriate fat to provide the essential fatty acids in rodent diets. Consequently, soy oil was included in the NTP-2000 diet to provide the essential and other fatty acids recommended by AIN. Corn oil was added to bring the fat level up to the planned amount to decrease or delay the incidence of leukemia in male F344 rats. Nutrients Nutrient concentrations obtained by analysis of 15 contemporary lots of NTP-2000 and NIH-07 diets are given in Table 3. Macronutrients. Protein is the source of amino acids necessary for growth and maintenance of rodents. Approximately 12% ideal protein was reported to be adequate for their growth (Reeves et al., 1993; National Research Council, 1995). Results of a 2-year study with 15% crude protein nonpurified diet supplemented with methionine indicated that 15% crude protein is adequate for growth and maintenance of rats (Rao et al., 1993). A higher dietary protein (23%) increased the severity of nephropathy. A 12% crude protein level was not considered to be adequate for growth and maintenance of rats subjected to stresses of chemical
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treatment in chemical toxicology studies (Rao, 1994). Accordingly, the NTP-2000 diet was formulated to contain approximately 14.5% protein from animal and cereal sources and was supplemented with methionine. Fat provides essential fatty acids for growth and maintenance. Soy oil at 3 to 5% in diet is expected to provide adequate amounts of linolenic and linolenic acids for growth and maintenance of rodents (Reeves et al., 1993). The soy oil content of NTP-2000 diet was 3%. The cereal ingredients and additional protein sources contributed Ç2.5% fat (Committee on Animal Nutrition, 1982) to this diet. In previous studies a high corn oil intake either as gavage or through diet appears to decrease or delay the development of leukemia a lethal tumor in male F344 rats (Rao and Haseman, 1993; NTP, 1994; Rao, 1994; Rao et al., 1996). Consequently, corn oil was added at 3% to increase the fat content of NTP2000 diet to approximately 8.5%. Increasing the fat content increases caloric density of diet which may increase caloric intake, body weight, and associated tumors (Freedman et al., 1990). To compensate for some of this effect, the crude fiber was increased.
TABLE 4 Vitamin and Mineral Concentrations of NIH-07 and NTP-2000 Nonpurified Diets with NRC 1995 Recommendations Nutrienta
NIH-07b
NTP-2000c
NRC
A (IU/kg) D (IU/kg) E Ke Thiamin Riboflavin Niacin Pantothenic Acid Pyridoxine Folic Acid Biotin B12 (ppb) Choline Sodium Potassium Calcium Phosphorus Magnesium Iron Manganese Copper Zinc Iodine Selenium
6300d 4450 37 3.0 20.7d 7.9 101 30 9.3 2.5 0.27 40 3070 3150 8870 11800d 9400d 1680 356 92 11.5 58 3.4 0.34d
6800d 1000e 84 1.0 12.1d 7.7 79 29 12.4 2.3 0.28 42 3200 1705 6590 9400d 5800d 1845 193 54 8.5 51 1.8 0.25d
2300 1000 18 1.0 4.0 3.0 15 10 6 1.0 0.2 50 750 500 3600 5000 3000 500 35 10 5 12 0.15 0.1
a
As ppm, unless stated in other units. Mean values of 10 lots analyzed during 1985–1995. c Results of one lot analyzed in 1995. d Mean values of 15 contemporary lots. e Added concentration, not analyzed. b
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TABLE 5 Selected Ultratrace Element Concentrations of Some Purified and Nonpurified Rodent Diets Purified AIN dietsa
Nonpurified dietsb
Element (ppm)
93G
93M
NIH-07
NTP-2000
Boron Chromium Fluoride Lithium Molybdenum Nickel Silicon Vanadium
0.5 1.0 1.0 0.1 0.18 0.5 1.0 0.1
0.5 1.0 1.0 0.1 0.17 0.5 1.0 0.1
7.4 1.1 13.1 1.0 0.84 2.5 1140 1.4
5.5 0.8 3.3 4.1 0.77 2.5 2200 0.8
a b
AIN (Reeves et al., 1993) and NRC (1995) recommendations. As determined by analysis of one contemporary lot.
No dietary fiber requirements for rodents has been established (National Research Council, 1995). Approximately 9.5% crude fiber has been formulated into this diet to decrease the caloric value (due to higher fat content) to be approximately the same as the NIH-07 diet. The adjustment of caloric value of NTP-2000 diet with fiber was necessary to maintain the amount of food consumed to Ç16 g/rat, so that the protein consumed will be adequate for growth and maintenance. Furthermore, increasing the fiber content helped to delay the development of mammary tumors in females and may have decreased the development of adrenal pheochromocytomas in male rats (Rao, 1994, 1995; Rao et al., 1996). However, increased fiber consumption may decrease intestinal transit time and decrease the availability of metabolizable energy (Miles, 1992; Rao et al., 1996). Calcium (Ca) and phosphorous (P) are essential minerals. The Ca:P ratio appears to be important to prevent nephrocalcinosis in female rats (Reeves et al., 1993). For this reason, in accordance with the AIN recommendation (Reeves et al., 1993), the Ca and P levels of this diet were adjusted to have a Ca:P molar ratio of greater than 1 and less than 2. Micronutrients. Vitamin concentrations in NTP-2000 diet in comparison with NIH-07 diet and NRC-95 recommendations are listed in Table 4. Nonpurified ingredients contribute variable amounts of vitamins such as A, E, K, and B-complex. Dried brewer’s yeast is a good source of B-vitamins. However, bioavailability of these vitamins is unknown. To assure that adequate vitamin levels for growth and maintenance are present in this diet, levels recommended (Reeves et al., 1993; National Research Council, 1995) to meet the dietary requirements were added to the formulation (Table 2). Mineral concentrations of NTP-2000 diet along with NIH07 diet and NRC-95 recommendations are given in Table 4.
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Trace minerals are present in various nonpurified ingredients used in the NTP-2000 diet, but the bioavailability of these minerals is not known. To assure adequacy of these trace minerals, recommended levels (Reeves et al., 1993; National Research Council, 1995) were added to this diet (Table 2). Both AIN (Reeves et al., 1993) and NRC (1995) recommended various ultratrace elements such as chromium, boron, fluoride, nickel, lithium, vanadium, molybdenum, and silicon as potentially beneficial elements for rodents. Ultratrace element concentrations in nonpurified diets will be more than adequate. One exception could be the chromium level. Molasses contributes chromium to the diet. Since no molasses was used in the NTP-2000 formulation, chromium was added (Table 2). Ultratrace element concentrations of NTP-2000 and NIH-07 diets as determined by analysis along with recommended concentrations in AIN (Reeves et al., 1993) diets are given in Table 5. The NTP-2000 diet is an open formula diet (American Institute of Nutrition, 1977) and details of the formulation are given in Tables 1 and 2. Any facility with experience in manufacturing rodent diets may make this diet. The contaminant limits for the NTP-2000 diet are the same as those recommended for the NIH-07 diet (Rao and Knapka, 1987). Commonly detected contaminant concentrations in 15 contemporary lots of NTP-2000 and NIH-07 diets are given in Table 6. The concentrations of most heavy metals were generally lower with significantly (p õ 0.001) lower concentrations of arsenic and nitrosamines in the NTP-2000 diet than in the NIH-07 diet due mostly to lower amount of fish meal (4% vs 10%) in the NTP-2000 diet. RAT STUDY
An experimental diet (NTP-90) with similar composition as the NTP-2000 diet was evaluated by a 2-year study and
TABLE 6 Contaminant Concentrations of NTP-2000 and NIH-07 Nonpurified Dietsa Contaminant
NTP-2000
NIH-07
Arsenic (ppm) Cadmium (ppm) Lead (ppm) Mercury (ppm) Selenium (ppm) Aflatoxins (ppb) Total volatile nitrosamines (ppb) N-Nitrosodimethylamine (ppb)
0.32 { 0.07** 0.05 { 0.02 0.23 { 0.07 õ0.02 0.25 { 0.15* õ5 7.85 { 2.53** 5.57 { 1.86**
0.65 { 0.13 0.06 { 0.03 0.28 { 0.12 õ0.02 0.34 { 0.09 õ5 11.43 { 3.08 9.47 { 3.17
a
Mean { standard deviation of common contaminants in 15 contemporary lots. * p õ 0.05. ** p õ 0.001.
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GHANTA N. RAO throughout the study. Water consumptions for a one-week period were determined at the 4th, 8th, and 12th weeks. Blood samples were obtained by orbital sinus bleeding under CO2:O2 (70:30) anesthesia from 10 rats of each sex and group during the 12th week and evaluated for hematology and blood chemistry parameters. During the 14th week of the study the rats were euthanatized with CO2 and necropsied. At necropsy liver, heart, kidney, gonads, brain, pituitary, adrenals, and thyroid were weighed, and all gross lesions and major organs/tissues were collected and fixed in 10% neutral buffered formalin. Lungs, liver, heart, spleen, kidneys, pancreas, gonads, brain, pituitary, thyroid, adrenals, and any gross lesions were processed, embedded in paraffin, and sectioned at 5 mM. Sections stained with hematoxylin and eosin were evaluated by light microscopy. Nephropathy and cardiomyopathy were evaluated for severity according to the criteria described by Dixon et al. (1995).
RESULTS AND DISCUSSION FIG. 1. Growth patterns of male and female Fischer 344 rats fed NIH07 (l) or NTP-2000 (s) diets for 13 weeks. q 1996 Society of Toxicology
reported elsewhere (Rao, 1995; Rao et al., 1996). The NTP2000 diet was evaluated by a 13-week study with the same protocol as the NTP 13-week chemical toxicity studies to document the growth patterns, palatability of diet, food consumption, clinical chemistry parameters and pathologic changes in F344 rats when fed NTP-2000 diet in comparison with the NIH-07 diet. MATERIALS AND METHODS Fischer-344 rats produced in a pathogen-free NTP production colony at Taconic Farms (Germantown, NY), were received at NIEHS when they were 4 weeks of age. Animals and animal care procedures were described by us elsewhere (Rao et al., 1993, 1996). Groups of 25 rats of both sexes were fed the NTP-2000 diet or the NIH-07 diet (both diets were manufactured by Zeigler Bros. Inc., Gardners, PA) for 13-weeks. The study was conducted in a facility of the NIEHS, accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC, Rockville, MD), with a protocol approved by the NIEHS Animal Care and Use Committee. Rats were housed five per cage by sex in hanging-drawer-type polycarbonate cages with heat-treated hardwood bedding as the contact bedding. Observations. The rats were examined daily and individual body weights were obtained weekly. Food consumptions by cage were measured
Growth curves for male and female rats fed both diets are shown in Fig. 1. Growth patterns were essentially the same for both diets except the male rats fed NTP-2000 diet had slower growth rate during first 6 weeks of the study. Final body weights, and average food and water consumptions were given in Table 7. Final body weights were the same for both diets. The NTP-2000 diet consumption was 5–10% higher (p õ 0.01) than the NIH-07 diet. Higher food consumption may be due to higher fiber content of this diet, even though the caloric (physiological fuel) value of NTP2000 diet was the same or slightly higher than the NIH-07 diet (Table 3). Furthermore, due to higher fiber content, the pellets of the NTP-2000 diet were not as hard as the NIH07 diet and this may have increased the proportion of diet lost in the bedding. A 2-year study with an experimental diet (NTP-90) with similar composition as the NTP-2000 diet did not effect the growth pattern for about 6 months but significantly (p õ 0.01) decreased the adult body weight, even though the food consumption of the experimental diet (NTP-90) group was Ç12% higher than the NIH-07 diet group (Rao et al., 1996). Water consumption of male rats fed NTP-2000 diet was lower (p õ 0.05) than the NIH-07 diet and this may be related to the decreased severity of nephropathy.
TABLE 7 Final Body Weights and Food and Water Consumption by F344 Rats Fed NIH-07 or NTP-2000 Nonpurified Diet for 13 Weeks Malea
Body weight (g)b Food (g/rat/day)c Water (g/rat/day)c
Femalea
NIH-07
NTP-2000
NIH-07
NTP-2000
319 { 24.8 16.0 { 0.30 20.0 { 0.80
318 { 19.8 16.9 { 0.20** 17.0 { 0.40*
188 { 14.4 10.6 { 0.16 14.2 { 0.50
189 { 12.5 11.6 { 0.11** 13.3 { 0.24
a
N Å 25. Mean { SD. c Mean { SE. * p õ 0.05. ** p õ 0.01. b
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TABLE 8 Serum Clinical Chemistry Parameters of F344 Rats Fed NIH-07 or NTP-2000 Nonpurified Diet for 11 Weeks Malea NIH-07 Total protein (g/dl) Albumin (g/dl) Urea nitrogen (mg/dl) Creatinine (mg/dl) Creatine kinase (IU/liter) Triglycerides (mg/dl) Cholesterol (mg/dl) Bile acids (umol/liter) Glucose (mg/dl) Alkaline phosphatase (IU/liter) 5* Nucleotidase (IU/liter) Alanine aminotransferase (IU/liter) Sorbitol dehydrogenase (IU/liter) Calcium (ppm) Phosphorus (ppm) Sodium (mmol/liter) Potassium (mmol/liter)
NTP-2000
Femalea Organ
07
2000
07
2000
Liver (g) Kidney (g) Adrenal (mg) Brain (g)
12.36 2.14 56 1.94
11.04** 1.94** 50** 1.92
6.00 1.26 56 1.72
5.36** 1.15** 52** 1.78**
7.29 4.12 21.8 0.89 163 325 76 22.5 133 177
7.11* 3.9* 13.5 0.91 120 261* 87** 20.7 137 186
7.12 4.19 21.7 0.85 133 228 110 20.8 125 132
7.44* 4.36** 16.4** 0.91 100 176 112 19.2 137 146*
26.9 43.1
30.4* 47.9
31.1 31.2
37.2** 45.7**
17.4
17.6
18.1
19.2
116 81 146 5.4
114 71* 147 5.2
118 57 144 4.8
118 54 144 4.8
There were no significant differences in hematological parameters of male or female rats fed the NTP-2000 or NIH-07 diets. Serum clinical chemistry parameters of groups fed both diets are listed in Table 8. There were slight (p õ 0.05) but not physiologically significant differences in serum protein and albumin concentrations. The blood urea nitrogen levels were markedly lower in male and female groups fed the NTP-2000 diet and this result was in agreement with our previous observations (Rao et al., 1996) with lower protein diets. The cholesterol level in males and 5* nucleotidase and alkaline phosphatase levels in both sexes fed the NTP-2000 diet were slightly higher possibly due to higher fat content of this diet (Kim et al., 1976; Young et al., 1981). However, the triglyceride levels in both sexes fed NTP-2000 diet were lower than the rats fed the NIH-07 diet, even though the fat content of NTP-2000 diet was higher than the NIH-07 diet and this difference may be due to higher fiber content of the NTP-2000 diet. The alanine aminotransferase concentration in females fed NTP-2000 diet was slightly higher (p õ 0.05) but within the range of historical control data (NTP unpublished data base). The serum calcium, phosphorus, sodium and potassium concentrations are the same for both diets except that the phosphorus levels of male rats fed NTP-2000 diet was slightly lower (p õ 0.01),
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NTP-2000
N Å 10. * p õ 0.05. ** p õ 0.01.
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NIH-07
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TABLE 9 Weights of Selected Organs of F344 Rats Fed NIH-07 or NTP-2000 Diet for 13 Weeksa
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a
There were no significant differences in heart, thyroid, pituitary, testis, ovary, and body weights. b N Å 25. ** p õ 0.01.
possibly due to lower phosphorus content of NTP-2000 diet (Table 3). Organ weights of male and female rats fed the two diets are given in Table 9. The liver and kidney weights of male and female rats fed NTP-2000 were significantly lower (p õ 0.01) than the group fed NIH-07 diet. The lower liver and kidney weights appears to be due to the lower protein content of NTP-2000 diet and may be related to lower protein consumption-associated changes in Phase I and Phase II drug metabolizing enzyme systems of liver and kidney (Butler and Dauterman, 1988). The adrenal weights of rats fed NTP-2000 diet were significantly lower (p õ 0.01) than the rats fed NIH-07 diet and the reason for this difference is not known. However, 2-year studies with experimental diets similar to NTP-2000 diet caused a significant decrease in the incidence of adrenal medullary hyperplasia and adrenal pheochromocytomas (Rao, 1995; Rao et al., 1996) and lower adrenal weights may be related to this effect. The most common lesions of F344 rats fed NIH-07 diet in 13week studies were nephrocalcinosis in females and initial changes of nephropathy and cardiomyopathy in both sexes
TABLE 10 Incidences of Selected Lesions in Fischer 344 Rats Fed NIH-07 or NTP-2000 Diet for 13 Weeks Malea
Femalea
Lesions (%)
07
2000
07
2000
Nephrocalcinosis Renal tubule regenerationb Cardiomyopathy
0 96 (1.0) 100
0 64 (0.6) 100c
96 0 64
0 4 52
a
N Å 25. Incidence % with (mean severity grade). c Less severe in the NTP-2000 fed group as determined by an independent pathologist. b
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(Dixon et al., 1995). Nephrocalcinosis was characterized microscopically by foci of crystalline or lamellated concretions at the junction of the inner and outer stripes of the outer medulla. Initial changes of nephropathy include randomly scattered foci of a few to several regenerative renal tubules with basophilic staining. Early changes of cardiomyopathy was characterized by randomly distributed focal to multifocal myofiber degeneration with mononuclear inflammatory cell infiltrates. The incidences of these lesions in male and female rats fed the two diets are given in Table 10. The NTP-2000 diet prevented nephrocalcinosis, decreased the incidence and severity of nephropathy in males, and decreased the severity of cardiomyopathy. There were no other significant lesions in rats fed either diet in the 13-week study. The NTP-2000 has not been tested for adequacy in supporting reproduction and lactation and this diet may not be appropriate or adequate for studies involving dietary/caloric restriction due to lower concentration of protein (Ç15%, which is adequate for growth when fed ad libitum) than the standard diets with higher concentrations of protein (18– 25%) routinely used for dietary/caloric restriction and reproduction and lactation. In conclusion, based on a 2-year study with an experimental diet (NTP-90) with similar composition as the NTP-2000 diet (Rao, 1995; Rao et al., 1996) and the results of the 13week study included in this report, the NTP-2000 diet is adequate for growth and maintenance of rats, prevented the development of nephrocalcinosis, and appears to decrease the incidence or severity of diet and possibly age-associated lesions in rats. ACKNOWLEDGMENTS The author acknowledges with thanks the generous assistance of Dr. Damon C. Shelton and Dr. Joseph J. Knapka in formulation of the NTP2000 diet and review of the manuscript, Dr. Gregory S. Travlos for assistance in interpretation of clinical chemistry data, and Dr. John Seely of Pathco for necropsy of rats and histopathologic evaluation of tissues including the severity of nephropathy and cardiomyopathy.
REFERENCES American Institute of Nutrition (1977). Report of the AIN Ad Hoc Committee on standards for nutritional studies. J. Nutr. 37, 1340–1348. Butler, L. E., and Dauterman, W. C. (1988). The effect of dietary protein levels on xenobiotic biotransformations in F344 male rats. Toxicol. Appl. Pharmacol. 95, 301–310. Committee on Animal Nutrition, National Research Council (1982). United
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States–Canadian Tables of Feed Composition. National Academy Press, Washington, DC. Dixon, D., Heider, K., and Elwell, M. R. (1995). Incidence of nonneoplastic lesions in historical control male and female Fischer-344 rats from 90-day toxicity studies. Toxicol. Pathol. 23, 338–348. FASEB Life Science Research Review Office (1987). Physiological Effects and Consequences of Dietary Fiber. FASEB, Bethesda, MD. Freedman, L. S., Clifford, C., and Messina, M. (1990). Analysis of dietary fat, calories, body weight and the development of mammary tumors in rats and mice: A review. Cancer Res. 50, 5710–5719. Kim, J. J., Hamilton, R. M. G., and Carroll, K. K. (1976). Effects of diet on catabolism and excretion of (26-C14) cholesterol in rats. Can. J. Biochem. 54, 272–279. Klahr, S., Buerkert, J., and Purkerson, M. L. (1983). Role of dietary factors in progression of renal disease. Kidney Int. 24, 579–587. Knapka, J. J., Smith, P. K., and Judge, F. J. (1974). Effect of open and closed formula rations on the performance of three strains of laboratory mice. Lab. Anim. Sci. 24, 480–487. Miles, C. W. (1992). The metabolizable energy of diets differing in dietary fat and fiber measured in humans. J. Nutr. 122, 306–311. Murphy, P. A. (1982). Phytoestrogen content of processed soybean products. Food Technol. 36, 60–64. National Research Council, Committee on Animal Nutrition (1995). Nutrient requirements of the laboratory rat. In Nutrient Requirements of Laboratory Animals, pp. 11–79. National Academy Press, Washington, DC. NTP (1994). Comparative Toxicology Studies of Corn Oil, Safflower Oil, and Tricaprylin in Male F344/N Rats as Vehicles for Gavage. NTP technical report 426, NIH Publication 94-3157. National Toxicology Program, Research Triangle Park, NC. Rao, G. N. (1994). Diet for Fischer-344 rats in long-term studies. Environ. Health Perspect. 102, 314–315. Rao, G. N. (1995). Husbandry procedures other than dietary restriction for lowering body weight and tumor/disease rates in Fischer 344 rats. In Dietary Restriction: Implications for the Design and Interpretation of Toxicity and Carcinogenicity Studies (D. A. Neumann, R. W. Hart, and R. T. Robertson, Eds.), pp. 51–62. ILSI Press, Washington, DC. Rao, G. N. (1991). Significance of dietary contaminants in diet restriction studies. In Biological Effects of Dietary Restriction (L. Fishbein, Ed.), pp. 16–24. Springer-Verlag, New York. Rao, G. N., Edmondson, J., and Elwell, M. R. (1993). Influence of dietary protein concentration on severity of nephropathy in Fischer-344 (F-344/ N) rats. Toxicol. Pathol. 21, 353–361. Rao, G. N., Edmondson, J., Hildebrandt, P. K., and Bruner, R. H. (1996). Influence of dietary protein, fat, and fiber on growth, blood chemistry and tumor incidences in Fischer 344 rats. Nutr. Cancer. 25, 269–279. Rao, G. N., and Haseman, J. K. (1993). Influence of corn oil and diet on body weight, survival, and tumor incidences in F344/N rats. Nutr. Cancer. 19, 21–30. Rao, G. N., and Knapka, J. J. (1987). Contaminant and nutrient concentrations of natural ingredient rat and mouse diet used in chemical toxicology studies. Fundam. Appl. Toxicol. 9, 329–338. Reeves, P. G., Nielsen, F. H., and Fahey, G. C., Jr. (1993). AIN-93 purified diets for laboratory rodents: Final report of the American Institute of Nutrition Ad Hoc writing committee on reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951. Young, G. B., Friedman, S., Yedlin, S. T., and Alpers, D. H. (1981). Effects of fat feeding on intestinal alkaline phosphatase activity in tissue and serum. Am. J. Physiol. 241, G461–G468.
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New Diet (NTP-2000) for Rats in the National Toxicology Program Toxicity and Carcinogenicity Studies GHANTA N. RAO National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received December 12, 1995; accepted March 30, 1996
New diet (NTP-2000) for Rats in the National Toxicology Program Toxicity and Carcinogenicity Studies. RAO, G. N. (1996). Fundam. Appl. Toxicol. 32, 102–108. Composition of diet may influence growth, diseases, tumor rates, and responses to chemical treatment. Since 1980 the NIH-07 open formula nonpurified diet has been the selected diet for the National Toxicology Program (NTP) toxicity and carcinogenicity studies in rodents. Studies with nonpurified experimental diets with lower protein and higher fat and fiber than the NIH-07 diet indicated that the diet for Fischer-344 (F344) rats in long-term studies could be modified to decrease the severity of chronic diseases and to decrease/delay the development of spontaneous tumors. Based on the results of these studies a new open formula nonpurified diet designated as NTP-2000 was formulated to contain Ç14.5% protein, Ç8.5% fat, and Ç9.5% fiber. Corn, wheat, and wheat middlings contribute to about 60% of the ingredients; soybean meal, fish meal, and alfalfa meal are the additional sources of protein; purified cellulose, oat hulls, and alfalfa meal are the major sources of fiber; and soy oil and corn oil are the major sources of fat in the NTP-2000 diet. The Ca:P ratio and mineral and vitamin concentrations were reformulated based on AIN-93 and NRC-95 recommendations. The NIH-07 and the NTP-2000 diets were fed to groups of 6-week-old F344 rats for 13 weeks and evaluated for growth patterns, food and water consumptions, hematology and clinical chemistry parameters, and organ weights and pathological changes. Growth patterns and body weights were similar for both diets. Food consumptions were slightly higher and water consumptions were slightly lower for the groups fed NTP-2000 diet. There were no differences in hematological parameters between the groups fed the above diets. Serum levels of cholesterol, alkaline phosphatase, and 5* nucleotidase were slightly higher in groups fed the NTP-2000 diet possibly due to higher fat content of this diet. However, the serum triglyceride levels were slightly lower in groups fed the NTP-2000 diet and it may be related to higher fiber content of the NTP-2000 diet. The liver and kidney weights of the groups fed NTP-2000 diet were significantly lower possibly due to lower protein content of this diet and lower protein consumption associated changes in Phase I and Phase II drug metabolizing enzyme systems. The adrenal weights were also lower in groups fed the new diet. The NTP-2000 diet prevented nephrocalcinosis and decreased the severity of nephropathy and cardiomyopathy, the common lesions of F344 rats in 13-week studies. These results indicate that the NTP-2000 diet is adequate for growth and main-
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Diet is an important factor influencing biological processes such as growth, survival, and diseases including tumor rates. Since 1980 the NIH-07 open formula nonpurified diet (Rao and Knapka, 1987) with high protein (Ç24%), low fat (Ç5%), and low fiber (Ç3.5%) has been the selected diet for the National Toxicology Program (NTP) sponsored chemical toxicity and carcinogenicity studies in rodents. This diet was formulated more than 20 years ago for reproduction, lactation, and growth of rodents (Knapka et al., 1974). Some components of NIH-07 diet such as high protein content may have increased the severity of nephropathy. Since 1988 a number of studies were conducted to obtain data on the influence of dietary protein, fat, and fiber (Rao et al., 1993; Rao and Haseman, 1993; NTP, 1994; Rao, 1994, 1995; Rao et al., 1996) on chronic diseases and tumor incidences. Decrease in protein consumption due to lower protein content of diet decreased the severity of nephropathy in rats without a significant effect on growth (Rao et al., 1993). In 2-year chemical carcinogenicity studies when corn oil was given by gavage (as a vehicle), there was a decrease in the incidences of leukemia, a lethal tumor in Fischer-344 (F344) rats. However, corn oil gavage significantly increased the adult body weight and pancreatic acinar cell tumors in male rats (Rao and Haseman, 1993; NTP, 1994). Higher fiber content of experimental diets appears to have delayed the development of mammary tumors in females (Rao, 1994, 1995; Rao et al., 1996). Higher fat or fiber of experimental diets decreased the incidences of adrenal medullary hyperplasia and adrenal pheochromocytomas in males (Rao, 1994, 1995; Rao et al., 1996). Lower protein and higher fat and fiber of experimental diets decreased the spontaneous tumor burden in 2-year studies (Rao et al., 1996). Results of these studies indicated that change in diet composition appears to be appropriate for rats in toxicity and carcinogenicity studies. A workshop was held at the National Institute of Environmental Health Sciences (NIEHS) in June 1993 to discuss possible changes in diet formulation and composition to de-
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TABLE 1 Ingredient Composition of NTP-20000 and NIH-07 Open Formula Nonpurified Diets Amount by weight (%) Ingredient
NTP-2000
NIH-07a
Ground wheat Ground corn Wheat middlings Soybean meal (49% protein) Fish meal (60% protein) Dried skim milk Corn gluten meal (60% protein) Dried brewer’s yeast Dry molasses Alfalfa meal (17% protein) Oat hulls Purified cellulose Corn oil (without preservatives) Soy oil (without preservatives) Sodium chloride Calcium phosphate, dibasic (USP) Calcium carbonate (USP) Choline chloride (70% choline) Methionine Vitamin premixc Mineral premixd
22.26 22.18 15.0 5.0 4.0 0.0 0.0 1.0 0.0 7.5 8.5 5.5 3.0 3.0 0.3 0.4 0.9 0.26 0.2 0.5 0.5
23.0 24.5 10.0 12.0 10.0 5.0 3.0 2.0 1.5 4.0 0.0 0.0 0.0 2.5 0.5 1.25 0.5 0.09b 0.0 0.125 0.125
nitrosamines, and chlorinated hydrocarbons (Rao, 1991). To decrease the contaminant levels of the new diet, limited amounts of these ingredients were used as protein sources. Additional protein from alfalfa meal and dried brewer’s yeast helped to complete the protein composition. Dried skim milk has casein as its major protein and casein may increase the severity of nephropathy in rats (Klahr et al., 1983). Furthermore, the availability of dried skim milk as an ingredient for laboratory animal diets is limited, so skim milk was not included in formulation of the new diet. Alfalfa meal is a good source of protein and fiber; oat hulls and beet pulp are good sources of fiber in experimental animal diets. Beet pulp was excluded since it has a high water-holding capacity and may disproportionately increase the bulk of intestinal contents (Rao, 1994). Alfalfa meal is a potential source of nitrosamines and oat hulls reduce pellet quality. High amounts of these ingredients may add high levels of phytates which may interfere with mineral absorption (FASEB, 1987). Thus, the proportion of either ingredient was less than 10% in the NTP-2000 diet. Approximately 50% of the crude fiber was from the above ingredients. Purified cellulose with the same specifications as the AIN93 diet (Reeves et al., 1993) was added to bring the crude fiber level up to the planned amount. The cereal ingredients and additional protein sources con-
a
Vitamin and mineral premix composition was given by AIN (1977). Included in the vitamin premix. c Wheat middlings as carrier. d Calcium carbonate as carrier. b
crease the severity of chronic diseases and to delay the development of spontaneous tumors in 2-year studies (Rao, 1994). A new diet designated as NTP-2000 has evolved from the above-mentioned efforts. This report provides (a) ingredient and nutrient composition of the new diet with explanations for their selection and (b) results of a 13-week study in F344 rats with the new diet. DIET COMPOSITION
Ingredients The ingredient composition of the NTP-2000 diet in comparison with the NIH-07 diet is provided in Table 1. Details of vitamin and mineral additions to NTP-2000 diet are listed in Table 2. This is a cereal-based nonpurified diet (American Institute of Nutrition, 1977) with corn, wheat, and wheat middling contributing to about 60% of the ingredients. Protein sources such as soybean meal, fish meal, and dried skim milk are commonly used in laboratory animal diets. Soybean products contribute to estrogenic activity due to naturally occurring phytoestrogens (Murphy, 1982). Fish meal can be a major source of contaminants such as heavy metals,
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TABLE 2 Vitamins and Minerals Added to the NTP-2000 Open Formula Nonpurified Dieta
Vitamins A D K a-Tocopheryl acetate Folic acid Niacin d-pantothenic acid Riboflavin Thiamin B12 Pyridoxine Biotin Minerals Magnesium Iron Zinc Manganese Copper Iodine Chromium a b
Amount
Source
4000 IU
Stabilized vitamin A Palmitate or acetate D activated animal sterol Menadione (MSBC)b
1000 IU 1 100 1.1 2.3 10 3.3 4 52 mg 6.3 0.2 514 35 12 10 2 0.2 0.2
d-Calcium pantothenate Thiamine mononitrate Pyridoxine hydrochloride d-biotin Magnesium oxide Iron sulfate Zinc oxide Manganese oxide Copper sulfate Calcium iodate Chromium acetate
mg/kg of finished diet. MSBC Menadione sodium bisulfite complex.
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TABLE 3 Proximate and Other Selected Nutrient Composition of NTP-2000 and NIH-07 Open Formula Nonpurified Diets % By weighta Nutrient
NTP-2000
NIH-07
Protein Fat Fiber Calcium Phosphorus Vitamin A (IU/kg) Thiamin (ppm) Carbohydrateb Energy (kcal/g)c Energy (kcal/g)d
14.6 { 0.91 8.2 { 0.5 9.3 { 0.81 0.94 { 0.06 0.58 { 0.04 6800 { 1500 12.1 { 1.45 52 3.77 3.40
22.9 { 0.44 5.4 { 0.26 3.4 { 0.39 1.18 { 0.07 0.94 { 0.04 6300 { 600 20.7 { 3.2 51 3.58 3.44
a
Mean { standard deviation of 15 contemporary lots. Other than crude fiber, calculated by difference including the correction for moisture and ash. c Physiological fuel value. d Physiological fuel value with no caloric value for fiber. b
tribute fat to this diet. Soy oil, corn oil, and stabilized animal fats are the common sources of added fat in laboratory animal diets. Corn oil was the most common fat source in the previous NTP studies (Rao and Haseman, 1993; NTP, 1994; Rao, 1994, 1995; Rao et al., 1996) and was found to decrease or delay the development of leukemia in male F344 rats. Soy oil was recommended by the American Institute of Nutrition (AIN), (Reeves et al., 1993) as the most appropriate fat to provide the essential fatty acids in rodent diets. Consequently, soy oil was included in the NTP-2000 diet to provide the essential and other fatty acids recommended by AIN. Corn oil was added to bring the fat level up to the planned amount to decrease or delay the incidence of leukemia in male F344 rats. Nutrients Nutrient concentrations obtained by analysis of 15 contemporary lots of NTP-2000 and NIH-07 diets are given in Table 3. Macronutrients. Protein is the source of amino acids necessary for growth and maintenance of rodents. Approximately 12% ideal protein was reported to be adequate for their growth (Reeves et al., 1993; National Research Council, 1995). Results of a 2-year study with 15% crude protein nonpurified diet supplemented with methionine indicated that 15% crude protein is adequate for growth and maintenance of rats (Rao et al., 1993). A higher dietary protein (23%) increased the severity of nephropathy. A 12% crude protein level was not considered to be adequate for growth and maintenance of rats subjected to stresses of chemical
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treatment in chemical toxicology studies (Rao, 1994). Accordingly, the NTP-2000 diet was formulated to contain approximately 14.5% protein from animal and cereal sources and was supplemented with methionine. Fat provides essential fatty acids for growth and maintenance. Soy oil at 3 to 5% in diet is expected to provide adequate amounts of linolenic and linolenic acids for growth and maintenance of rodents (Reeves et al., 1993). The soy oil content of NTP-2000 diet was 3%. The cereal ingredients and additional protein sources contributed Ç2.5% fat (Committee on Animal Nutrition, 1982) to this diet. In previous studies a high corn oil intake either as gavage or through diet appears to decrease or delay the development of leukemia a lethal tumor in male F344 rats (Rao and Haseman, 1993; NTP, 1994; Rao, 1994; Rao et al., 1996). Consequently, corn oil was added at 3% to increase the fat content of NTP2000 diet to approximately 8.5%. Increasing the fat content increases caloric density of diet which may increase caloric intake, body weight, and associated tumors (Freedman et al., 1990). To compensate for some of this effect, the crude fiber was increased.
TABLE 4 Vitamin and Mineral Concentrations of NIH-07 and NTP-2000 Nonpurified Diets with NRC 1995 Recommendations Nutrienta
NIH-07b
NTP-2000c
NRC
A (IU/kg) D (IU/kg) E Ke Thiamin Riboflavin Niacin Pantothenic Acid Pyridoxine Folic Acid Biotin B12 (ppb) Choline Sodium Potassium Calcium Phosphorus Magnesium Iron Manganese Copper Zinc Iodine Selenium
6300d 4450 37 3.0 20.7d 7.9 101 30 9.3 2.5 0.27 40 3070 3150 8870 11800d 9400d 1680 356 92 11.5 58 3.4 0.34d
6800d 1000e 84 1.0 12.1d 7.7 79 29 12.4 2.3 0.28 42 3200 1705 6590 9400d 5800d 1845 193 54 8.5 51 1.8 0.25d
2300 1000 18 1.0 4.0 3.0 15 10 6 1.0 0.2 50 750 500 3600 5000 3000 500 35 10 5 12 0.15 0.1
a
As ppm, unless stated in other units. Mean values of 10 lots analyzed during 1985–1995. c Results of one lot analyzed in 1995. d Mean values of 15 contemporary lots. e Added concentration, not analyzed. b
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TABLE 5 Selected Ultratrace Element Concentrations of Some Purified and Nonpurified Rodent Diets Purified AIN dietsa
Nonpurified dietsb
Element (ppm)
93G
93M
NIH-07
NTP-2000
Boron Chromium Fluoride Lithium Molybdenum Nickel Silicon Vanadium
0.5 1.0 1.0 0.1 0.18 0.5 1.0 0.1
0.5 1.0 1.0 0.1 0.17 0.5 1.0 0.1
7.4 1.1 13.1 1.0 0.84 2.5 1140 1.4
5.5 0.8 3.3 4.1 0.77 2.5 2200 0.8
a b
AIN (Reeves et al., 1993) and NRC (1995) recommendations. As determined by analysis of one contemporary lot.
No dietary fiber requirements for rodents has been established (National Research Council, 1995). Approximately 9.5% crude fiber has been formulated into this diet to decrease the caloric value (due to higher fat content) to be approximately the same as the NIH-07 diet. The adjustment of caloric value of NTP-2000 diet with fiber was necessary to maintain the amount of food consumed to Ç16 g/rat, so that the protein consumed will be adequate for growth and maintenance. Furthermore, increasing the fiber content helped to delay the development of mammary tumors in females and may have decreased the development of adrenal pheochromocytomas in male rats (Rao, 1994, 1995; Rao et al., 1996). However, increased fiber consumption may decrease intestinal transit time and decrease the availability of metabolizable energy (Miles, 1992; Rao et al., 1996). Calcium (Ca) and phosphorous (P) are essential minerals. The Ca:P ratio appears to be important to prevent nephrocalcinosis in female rats (Reeves et al., 1993). For this reason, in accordance with the AIN recommendation (Reeves et al., 1993), the Ca and P levels of this diet were adjusted to have a Ca:P molar ratio of greater than 1 and less than 2. Micronutrients. Vitamin concentrations in NTP-2000 diet in comparison with NIH-07 diet and NRC-95 recommendations are listed in Table 4. Nonpurified ingredients contribute variable amounts of vitamins such as A, E, K, and B-complex. Dried brewer’s yeast is a good source of B-vitamins. However, bioavailability of these vitamins is unknown. To assure that adequate vitamin levels for growth and maintenance are present in this diet, levels recommended (Reeves et al., 1993; National Research Council, 1995) to meet the dietary requirements were added to the formulation (Table 2). Mineral concentrations of NTP-2000 diet along with NIH07 diet and NRC-95 recommendations are given in Table 4.
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Trace minerals are present in various nonpurified ingredients used in the NTP-2000 diet, but the bioavailability of these minerals is not known. To assure adequacy of these trace minerals, recommended levels (Reeves et al., 1993; National Research Council, 1995) were added to this diet (Table 2). Both AIN (Reeves et al., 1993) and NRC (1995) recommended various ultratrace elements such as chromium, boron, fluoride, nickel, lithium, vanadium, molybdenum, and silicon as potentially beneficial elements for rodents. Ultratrace element concentrations in nonpurified diets will be more than adequate. One exception could be the chromium level. Molasses contributes chromium to the diet. Since no molasses was used in the NTP-2000 formulation, chromium was added (Table 2). Ultratrace element concentrations of NTP-2000 and NIH-07 diets as determined by analysis along with recommended concentrations in AIN (Reeves et al., 1993) diets are given in Table 5. The NTP-2000 diet is an open formula diet (American Institute of Nutrition, 1977) and details of the formulation are given in Tables 1 and 2. Any facility with experience in manufacturing rodent diets may make this diet. The contaminant limits for the NTP-2000 diet are the same as those recommended for the NIH-07 diet (Rao and Knapka, 1987). Commonly detected contaminant concentrations in 15 contemporary lots of NTP-2000 and NIH-07 diets are given in Table 6. The concentrations of most heavy metals were generally lower with significantly (p õ 0.001) lower concentrations of arsenic and nitrosamines in the NTP-2000 diet than in the NIH-07 diet due mostly to lower amount of fish meal (4% vs 10%) in the NTP-2000 diet. RAT STUDY
An experimental diet (NTP-90) with similar composition as the NTP-2000 diet was evaluated by a 2-year study and
TABLE 6 Contaminant Concentrations of NTP-2000 and NIH-07 Nonpurified Dietsa Contaminant
NTP-2000
NIH-07
Arsenic (ppm) Cadmium (ppm) Lead (ppm) Mercury (ppm) Selenium (ppm) Aflatoxins (ppb) Total volatile nitrosamines (ppb) N-Nitrosodimethylamine (ppb)
0.32 { 0.07** 0.05 { 0.02 0.23 { 0.07 õ0.02 0.25 { 0.15* õ5 7.85 { 2.53** 5.57 { 1.86**
0.65 { 0.13 0.06 { 0.03 0.28 { 0.12 õ0.02 0.34 { 0.09 õ5 11.43 { 3.08 9.47 { 3.17
a
Mean { standard deviation of common contaminants in 15 contemporary lots. * p õ 0.05. ** p õ 0.001.
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GHANTA N. RAO throughout the study. Water consumptions for a one-week period were determined at the 4th, 8th, and 12th weeks. Blood samples were obtained by orbital sinus bleeding under CO2:O2 (70:30) anesthesia from 10 rats of each sex and group during the 12th week and evaluated for hematology and blood chemistry parameters. During the 14th week of the study the rats were euthanatized with CO2 and necropsied. At necropsy liver, heart, kidney, gonads, brain, pituitary, adrenals, and thyroid were weighed, and all gross lesions and major organs/tissues were collected and fixed in 10% neutral buffered formalin. Lungs, liver, heart, spleen, kidneys, pancreas, gonads, brain, pituitary, thyroid, adrenals, and any gross lesions were processed, embedded in paraffin, and sectioned at 5 mM. Sections stained with hematoxylin and eosin were evaluated by light microscopy. Nephropathy and cardiomyopathy were evaluated for severity according to the criteria described by Dixon et al. (1995).
RESULTS AND DISCUSSION FIG. 1. Growth patterns of male and female Fischer 344 rats fed NIH07 (l) or NTP-2000 (s) diets for 13 weeks. q 1996 Society of Toxicology
reported elsewhere (Rao, 1995; Rao et al., 1996). The NTP2000 diet was evaluated by a 13-week study with the same protocol as the NTP 13-week chemical toxicity studies to document the growth patterns, palatability of diet, food consumption, clinical chemistry parameters and pathologic changes in F344 rats when fed NTP-2000 diet in comparison with the NIH-07 diet. MATERIALS AND METHODS Fischer-344 rats produced in a pathogen-free NTP production colony at Taconic Farms (Germantown, NY), were received at NIEHS when they were 4 weeks of age. Animals and animal care procedures were described by us elsewhere (Rao et al., 1993, 1996). Groups of 25 rats of both sexes were fed the NTP-2000 diet or the NIH-07 diet (both diets were manufactured by Zeigler Bros. Inc., Gardners, PA) for 13-weeks. The study was conducted in a facility of the NIEHS, accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC, Rockville, MD), with a protocol approved by the NIEHS Animal Care and Use Committee. Rats were housed five per cage by sex in hanging-drawer-type polycarbonate cages with heat-treated hardwood bedding as the contact bedding. Observations. The rats were examined daily and individual body weights were obtained weekly. Food consumptions by cage were measured
Growth curves for male and female rats fed both diets are shown in Fig. 1. Growth patterns were essentially the same for both diets except the male rats fed NTP-2000 diet had slower growth rate during first 6 weeks of the study. Final body weights, and average food and water consumptions were given in Table 7. Final body weights were the same for both diets. The NTP-2000 diet consumption was 5–10% higher (p õ 0.01) than the NIH-07 diet. Higher food consumption may be due to higher fiber content of this diet, even though the caloric (physiological fuel) value of NTP2000 diet was the same or slightly higher than the NIH-07 diet (Table 3). Furthermore, due to higher fiber content, the pellets of the NTP-2000 diet were not as hard as the NIH07 diet and this may have increased the proportion of diet lost in the bedding. A 2-year study with an experimental diet (NTP-90) with similar composition as the NTP-2000 diet did not effect the growth pattern for about 6 months but significantly (p õ 0.01) decreased the adult body weight, even though the food consumption of the experimental diet (NTP-90) group was Ç12% higher than the NIH-07 diet group (Rao et al., 1996). Water consumption of male rats fed NTP-2000 diet was lower (p õ 0.05) than the NIH-07 diet and this may be related to the decreased severity of nephropathy.
TABLE 7 Final Body Weights and Food and Water Consumption by F344 Rats Fed NIH-07 or NTP-2000 Nonpurified Diet for 13 Weeks Malea
Body weight (g)b Food (g/rat/day)c Water (g/rat/day)c
Femalea
NIH-07
NTP-2000
NIH-07
NTP-2000
319 { 24.8 16.0 { 0.30 20.0 { 0.80
318 { 19.8 16.9 { 0.20** 17.0 { 0.40*
188 { 14.4 10.6 { 0.16 14.2 { 0.50
189 { 12.5 11.6 { 0.11** 13.3 { 0.24
a
N Å 25. Mean { SD. c Mean { SE. * p õ 0.05. ** p õ 0.01. b
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TABLE 8 Serum Clinical Chemistry Parameters of F344 Rats Fed NIH-07 or NTP-2000 Nonpurified Diet for 11 Weeks Malea NIH-07 Total protein (g/dl) Albumin (g/dl) Urea nitrogen (mg/dl) Creatinine (mg/dl) Creatine kinase (IU/liter) Triglycerides (mg/dl) Cholesterol (mg/dl) Bile acids (umol/liter) Glucose (mg/dl) Alkaline phosphatase (IU/liter) 5* Nucleotidase (IU/liter) Alanine aminotransferase (IU/liter) Sorbitol dehydrogenase (IU/liter) Calcium (ppm) Phosphorus (ppm) Sodium (mmol/liter) Potassium (mmol/liter)
NTP-2000
Femalea Organ
07
2000
07
2000
Liver (g) Kidney (g) Adrenal (mg) Brain (g)
12.36 2.14 56 1.94
11.04** 1.94** 50** 1.92
6.00 1.26 56 1.72
5.36** 1.15** 52** 1.78**
7.29 4.12 21.8 0.89 163 325 76 22.5 133 177
7.11* 3.9* 13.5 0.91 120 261* 87** 20.7 137 186
7.12 4.19 21.7 0.85 133 228 110 20.8 125 132
7.44* 4.36** 16.4** 0.91 100 176 112 19.2 137 146*
26.9 43.1
30.4* 47.9
31.1 31.2
37.2** 45.7**
17.4
17.6
18.1
19.2
116 81 146 5.4
114 71* 147 5.2
118 57 144 4.8
118 54 144 4.8
There were no significant differences in hematological parameters of male or female rats fed the NTP-2000 or NIH-07 diets. Serum clinical chemistry parameters of groups fed both diets are listed in Table 8. There were slight (p õ 0.05) but not physiologically significant differences in serum protein and albumin concentrations. The blood urea nitrogen levels were markedly lower in male and female groups fed the NTP-2000 diet and this result was in agreement with our previous observations (Rao et al., 1996) with lower protein diets. The cholesterol level in males and 5* nucleotidase and alkaline phosphatase levels in both sexes fed the NTP-2000 diet were slightly higher possibly due to higher fat content of this diet (Kim et al., 1976; Young et al., 1981). However, the triglyceride levels in both sexes fed NTP-2000 diet were lower than the rats fed the NIH-07 diet, even though the fat content of NTP-2000 diet was higher than the NIH-07 diet and this difference may be due to higher fiber content of the NTP-2000 diet. The alanine aminotransferase concentration in females fed NTP-2000 diet was slightly higher (p õ 0.05) but within the range of historical control data (NTP unpublished data base). The serum calcium, phosphorus, sodium and potassium concentrations are the same for both diets except that the phosphorus levels of male rats fed NTP-2000 diet was slightly lower (p õ 0.01),
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Femaleb
NTP-2000
N Å 10. * p õ 0.05. ** p õ 0.01.
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TABLE 9 Weights of Selected Organs of F344 Rats Fed NIH-07 or NTP-2000 Diet for 13 Weeksa
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a
There were no significant differences in heart, thyroid, pituitary, testis, ovary, and body weights. b N Å 25. ** p õ 0.01.
possibly due to lower phosphorus content of NTP-2000 diet (Table 3). Organ weights of male and female rats fed the two diets are given in Table 9. The liver and kidney weights of male and female rats fed NTP-2000 were significantly lower (p õ 0.01) than the group fed NIH-07 diet. The lower liver and kidney weights appears to be due to the lower protein content of NTP-2000 diet and may be related to lower protein consumption-associated changes in Phase I and Phase II drug metabolizing enzyme systems of liver and kidney (Butler and Dauterman, 1988). The adrenal weights of rats fed NTP-2000 diet were significantly lower (p õ 0.01) than the rats fed NIH-07 diet and the reason for this difference is not known. However, 2-year studies with experimental diets similar to NTP-2000 diet caused a significant decrease in the incidence of adrenal medullary hyperplasia and adrenal pheochromocytomas (Rao, 1995; Rao et al., 1996) and lower adrenal weights may be related to this effect. The most common lesions of F344 rats fed NIH-07 diet in 13week studies were nephrocalcinosis in females and initial changes of nephropathy and cardiomyopathy in both sexes
TABLE 10 Incidences of Selected Lesions in Fischer 344 Rats Fed NIH-07 or NTP-2000 Diet for 13 Weeks Malea
Femalea
Lesions (%)
07
2000
07
2000
Nephrocalcinosis Renal tubule regenerationb Cardiomyopathy
0 96 (1.0) 100
0 64 (0.6) 100c
96 0 64
0 4 52
a
N Å 25. Incidence % with (mean severity grade). c Less severe in the NTP-2000 fed group as determined by an independent pathologist. b
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(Dixon et al., 1995). Nephrocalcinosis was characterized microscopically by foci of crystalline or lamellated concretions at the junction of the inner and outer stripes of the outer medulla. Initial changes of nephropathy include randomly scattered foci of a few to several regenerative renal tubules with basophilic staining. Early changes of cardiomyopathy was characterized by randomly distributed focal to multifocal myofiber degeneration with mononuclear inflammatory cell infiltrates. The incidences of these lesions in male and female rats fed the two diets are given in Table 10. The NTP-2000 diet prevented nephrocalcinosis, decreased the incidence and severity of nephropathy in males, and decreased the severity of cardiomyopathy. There were no other significant lesions in rats fed either diet in the 13-week study. The NTP-2000 has not been tested for adequacy in supporting reproduction and lactation and this diet may not be appropriate or adequate for studies involving dietary/caloric restriction due to lower concentration of protein (Ç15%, which is adequate for growth when fed ad libitum) than the standard diets with higher concentrations of protein (18– 25%) routinely used for dietary/caloric restriction and reproduction and lactation. In conclusion, based on a 2-year study with an experimental diet (NTP-90) with similar composition as the NTP-2000 diet (Rao, 1995; Rao et al., 1996) and the results of the 13week study included in this report, the NTP-2000 diet is adequate for growth and maintenance of rats, prevented the development of nephrocalcinosis, and appears to decrease the incidence or severity of diet and possibly age-associated lesions in rats. ACKNOWLEDGMENTS The author acknowledges with thanks the generous assistance of Dr. Damon C. Shelton and Dr. Joseph J. Knapka in formulation of the NTP2000 diet and review of the manuscript, Dr. Gregory S. Travlos for assistance in interpretation of clinical chemistry data, and Dr. John Seely of Pathco for necropsy of rats and histopathologic evaluation of tissues including the severity of nephropathy and cardiomyopathy.
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