* 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 27709
Received March 20, 2001; accepted July 17, 2001
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ABSTRACT |
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Key Words: diet; nephropathy; cardiomyopathy; body weight; survival; group housing; individual housing; Fischer 344 rat.
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INTRODUCTION |
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The purpose of this report is to summarize the differences in 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.
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MATERIALS AND METHODS |
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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.
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Lesions.
Nephropathy and cardiomyopathy are the most common non-neoplastic 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. 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.
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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 = 0.05. 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 (Steel 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 (Steel 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.
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RESULTS |
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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 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.
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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 2.62 ± 0.056 (mean ± SE) for the NIH-07 diet and 1.86 ± 0.040 for the NTP-2000 diet groups. The severity was significantly (p < 0.001) lower in the NTP-2000 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 92% 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 1.44 ± 0.044 for the NIH-07 diet and 1.13 ± 0.025 for the NTP-2000 diet groups. The severity was significantly (p < 0.001) lower in the NTP-2000 diet groups.
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DISCUSSION |
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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-2000 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-2000 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-2000 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-2000-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 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-2000 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.
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ACKNOWLEDGMENTS |
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NOTES |
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