The effect of study type on body weight and tumor incidence in B6C3F1 mice fed the NTP-2000 diet

The B6C3F1 mouse is the standard mouse strain used in National Toxicology Program (NTP) carcinogenesis studies. Over time, increased liver tumorigenesis that was correlated with elevated body weights was noted in males and females. NTP therefore replaced the NIH-07 diet with the NTP-2000 diet and returned to group housing of females as lower body weights were noted in group housed mice. However, recent studies reported study-type differences in body weights at 3 months using the NTP-2000 diet with higher weights evident in drinking water and inhalation studies compared to feed studies. Therefore, body weight and tumor incidence data were collected for untreated control mice from all 2-year NTP feed (12), drinking water (8), water gavage (6) and inhalation (10) studies that used the NTP-2000 diet in order to assess the impact of study type on body weights and tumor incidences. Results show statistically significant elevated body weights and liver tumor incidences in males and females from drinking water, water gavage and inhalation studies compared to results from feed studies. Thus, the elevated body weights and liver tumorigenesis noted in mice using the NIH-07 diet were also evident using the NTP-2000 diet, which was introduced to address body weight elevations. Given the study-type dependent effects noted, these results emphasize the importance of carefully selecting historical control data for B6C3F1 mice. Moreover, because of the association between body weight and liver tumorigenesis, these results may have implications regarding dose-level selection for carcinogenicity studies involving B6C3F1 mice based on the maximum tolerated dose.     

The effect of study type on body weight and tumor incidence in B6C3F1 mice fed the NTP-2000 diet

The B6C3F1 mouse is the standard mouse strain used in National Toxicology Program (NTP) carcinogenesis studies. Over time, increased liver tumorigenesis that was correlated with elevated body weights was noted in males and females. NTP therefore replaced the NIH-07 diet with the NTP-2000 diet and returned to group housing of females as lower body weights were noted in group housed mice. However, recent studies reported study-type differences in body weights at 3 months using the NTP-2000 diet with higher weights evident in drinking water and inhalation studies compared to feed studies. Therefore, body weight and tumor incidence data were collected for untreated control mice from all 2-year NTP feed (12), drinking water (8), water gavage (6) and inhalation (10) studies that used the NTP-2000 diet in order to assess the impact of study type on body weights and tumor incidences. Results show statistically significant elevated body weights and liver tumor incidences in males and females from drinking water, water gavage and inhalation studies compared to results from feed studies. Thus, the elevated body weights and liver tumorigenesis noted in mice using the NIH-07 diet were also evident using the NTP-2000 diet, which was introduced to address body weight elevations. Given the study-type dependent effects noted, these results emphasize the importance of carefully selecting historical control data for B6C3F1 mice. Moreover, because of the association between body weight and liver tumorigenesis, these results may have implications regarding dose-level selection for carcinogenicity studies involving B6C3F1 mice based on the maximum tolerated dose.     

A 90-Day Chloroform Inhalation Study in F-344 Rats: Profile of Toxicity and Relevance to Cancer Studies

Composition of diet may influence growth, diseases, tumor rates,and responses to chemical treatment. Since 1980 the NIH–07open formula nonpurified diet has been the selected diet forthe National Toxicology Program (NTP) toxicity and carcinogenicitystudies in rodents. Studies with nonpurified experimental dietswith lower protein and higher fat and fiber than the NIH-07diet indicated that the diet for Fischer-344 (F344) rats inlong-term studies could be modified to decrease the severityof chronic diseases and to decrease/delay the development ofspontaneous tumors. Based on the results of these studies anew open formula nonpurified diet designated as NTP-2000 wasformulated to contain 14.5% protein, 8.5% fat, and 9.5% fiber.Corn, wheat, and wheat middlings contribute to about 60% ofthe ingredients; soybean meal, fish meal, and alfalfa meal arethe additional sources of protein; purified cellulose, oat hulls,and alfalfa meal are the major sources of fiber; and soy oiland corn oil are the major sources of fat in the NTP-2000 diet.The Ca:P ratio and mineral and vitamin concentrations were reformulatedbased on AIN-93 and NRC-95 recommendations. The NIH-07 and theNTP-2000 diets were fed to groups of 6-week-old F344 rats for13 weeks and evaluated for growth patterns, food and water consumptions,hematology and clinical chemistry parameters, and organ weightsand pathological changes. Growth patterns and body weights weresimilar for both diets. Food consumptions were slightly higherand water consumptions were slightly lower for the groups fedNTP-2000 diet. There were no differences in hematological parametersbetween the groups fed the above diets. Serum levels of cholesterol,alkaline phosphatase, and 5' nucleotidase were slightly higherin groups fed the NTP–2000 diet possibly due to higherfat content of this diet. However, the serum triglyceride levelswere slightly lower in groups fed the NTP–2000 diet andit may be related to higher fiber content of the NTP–2000diet. The liver and kidney weights of the groups fed NTP–2000diet were significantly lower possibly due to lower proteincontent of this diet and lower protein consumption associatedchanges 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 decreasedthe severity of nephropathy and cardiomyopathy, the common lesionsof F344 rats in 13–week studies. These results indicatethat the NTP–2000 diet is adequate for growth and maintenance of rats and appears to prevent or decrease the severityof diet-associated lesions.     

New Diet (NTP-2000) for Rats in the National Toxicology Program Toxicity and Carcinogenicity Studies

Composition of diet may influence growth, diseases, tumor rates,and responses to chemical treatment. Since 1980 the NIH–07open formula nonpurified diet has been the selected diet forthe National Toxicology Program (NTP) toxicity and carcinogenicitystudies in rodents. Studies with nonpurified experimental dietswith lower protein and higher fat and fiber than the NIH-07diet indicated that the diet for Fischer–344 (F344) ratsin long-term studies could be modified to decrease the severityof chronic diseases and to decrease/delay the development ofspontaneous tumors. Based on the results of these studies anew open formula nonpurified diet designated as NTP-2000 wasformulated to contain 14.5% protein, 8.5% fat, and 9.5% fiber.Corn, wheat, and wheat middlings contribute to about 60% ofthe ingredients; soybean meal, fish meal, and alfalfa meal arethe additional sources of protein; purified cellulose, oat hulls,and alfalfa meal are the major sources of fiber; and soy oiland corn oil are the major sources of fat in the NTP–2000diet. The Ca:P ratio and mineral and vitamin concentrationswere reformulated based on AIN–93 and NRC–95 recommendations.The NIH-07 and the NTP–2000 diets were fed to groups of6–week–old F344 rats for 13 weeks and evaluatedfor growth patterns, food and water consumptions, hematologyand clinical chemistry parameters, and organ weights and pathologicalchanges. Growth patterns and body weights were similar for bothdiets. Food consumptions were slightly higher and water consumptionswere slightly lower for the groups fed NTP–2000 diet.There were no differences in hematological parameters betweenthe groups fed the above diets. Serum levels of cholesterol,alkaline phosphatase, and 5' nucleotidase were slightly higherin groups fed the NTP–2000 diet possibly due to higherfat content of this diet. However, the serum triglyceride levelswere slightly lower in groups fed the NTP–2000 diet andit may be related to higher fiber content of the NTP–2000diet. The liver and kidney weights of the groups fed NTP-2000diet were significantly lower possibly due to lower proteincontent of this diet and lower protein consumption associatedchanges 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 decreasedthe severity of nephropathy and cardiomyopathy, the common lesionsof F344 rats in 13–week studies. These results indicatethat the NTP–2000 diet is adequate for growth and maintenance of rats and appears to prevent or decrease the severityof diet-associated lesions.     

The effect of chronic ingestion of lead on gastrointestinal transit in rats

GI symptoms such as constipation and abdominal colic are signs of lead poisoning in man, but mechanisms of these effects have not been elucidated. To evaluate GI transit, male Wistar rats were dosed with 1% lead or 0.7% sodium acetate in their diet (AIN-76A). After 7 weeks, lead-treated animals exhibited decreased hematocrit, increased 24-hr urinary excretion of delta-ALA, increased kidney/body weight ratio, and decreased body weight. Blood-lead concentrations were elevated to 196 +/- 57 micrograms/dl. Lead treatment, however, did not result in change in GI transit of a nonabsorbable marker, 51Cr, 15 min or 6 hr after po administration. There was also no change in fecal percentage water content. Since in control animals the semipurified diet AIN-76A markedly decreased fecal excretion rate of 51Cr compared to a cereal-based diet, NIH-07, the latter was used in subsequent experiments. Rats fed 2 or 4% lead acetate in NIH-07 for 8 weeks exhibited renal and hematologic toxicity as in the initial experiment. Weight gain was impaired in the 4% group compared to pair-fed controls. No significant differences were observed in the 1-hr gastric emptying or the fecal excretion of 51Cr in the 2 or 4% lead-treated animals, although there was a trend for slower transit in rats receiving the higher dose. These observations indicate that concentrations of lead sufficient to induce renal and hematologic toxicity in rats do not substantially affect GI transit.     

Absolute and relative organ weight trends in B6C3F1 mice

Since the early 1970s, the National Cancer Institute (NCI) and National Toxicology Program (NTP) have conducted carcinogenesis and toxicology studies on several hundred chemicals using the B6C3F1 mouse. A number of publications have examined growth, survival, and tumor incidence over time, including the impact of changes in housing and diet. However, no reports have been published to date examining the variation in organ weights over time, especially in light of reported body weight effects associated with housing and diet changes. Therefore, all available absolute and relative organ weight data for untreated control B6C3F1 mice were collected from 2-wk, 3-mo, and 15-mo NCI/NTP investigations with report dates through August 2010 in order to examine organ weight changes over time and by study type. Study data were grouped into 5-yr intervals by initiation date. Body weights in males increased over time except in 2-wk studies, while body weights in females rose through 1993 and remained constant or declined thereafter. Higher body weights were noted in individually housed mice, and in drinking water studies compared to feed or inhalation studies. Elevated organ weights were typically associated with increased body weights except that lower organ weights were evident as early as 2-wk on study with the introduction of the NTP-2000 diet in 1994. Relative organ weights decreased over time in males and females. Finally, organ weight coefficients of variation (standard deviation/mean) declined over time in 2-wk, 3-mo, and 15-mo studies, which may reflect improved data collection methods or reduced interlaboratory variability.     

Influence of dietary pectin on intestinal microfloral metabolism and toxicity of nitrobenzene

Intestinal microfloral metabolism of nitrobenzene is essential for the production of methemoglobin. Since dietary pectin alters intestinal microflora, these studies were designed to examine the effects of dietary pectin on nitrobenzene-induced methemoglobinemia. Male Fischer-344 rats were fed either AIN-76A (purified diet containing 5% cellulose), AIN-76A with 5% pectin replacing the cellulose, or NIH-07 (cereal-based diet containing 8.4% pectin) for 28 days. Following this period, nitrobenzene (200 mg/kg) was administered by gastric intubation, and methemoglobin concentrations were determined after 1, 2, 4, 8, and 24 hr. Nitrobenzene-induced methemoglobinemia was evident as early as 1 hr, peaked at 4 hr, and diminished thereafter in rats fed NIH-07 diet. In contrast, nitrobenzene-induced methemoglobinemia was not detectable in rats fed AIN-76A; however, inclusion of 5% pectin in this diet resulted in methemoglobinemia comparable to that of NIH-07-fed animals at 4, 8, and 24 hr. Administration of 400 or 600 mg/kg nitrobenzene resulted in significant diet-related differences in methemoglobinemia. Administration of 600 mg/kg nitrobenzene to animals fed NIH-07 resulted in the highest methemoglobin concentrations (64 ± 1%); those fed AIN-76A had the lowest (20 ± 5%), and those fed AIN-76A containing pectin had intermediate methemoglobin concentrations (44 ± 6%). No diet-related differences in the microbial population of the stomach or small intestine were observed. However, the number of anaerobes present in the ceca of rats fed AIN-76A containing pectin was 2 to 2.5 times greater than that of rats fed AIN-76A. In vitro reductive metabolism of [¹⁴C]nitrobenzene was significantly greater in the cecal contents of rats fed NIH-07 than that in the cecal contents of either of the groups fed the AIN-76A-based diets. These studies indicate that intestinal microfloral metabolism and red blood cell toxicity of nitrobenzene is markedly different in animals fed cereal-based versus purified diets. Furthermore, since inclusion of pectin into the purified diet diminishes the magnitude of these effects, differences in dietary composition of fermentable carbohydrates in cereal-based and purified diets may mediate differences in metabolism and toxicity of nitrobenzene.     

Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro.

Estrogenic isoflavones, such as genistein and daidzein, are present in virtually all natural-ingredient rodent diets that use soy as a source of protein. Since these compounds are endocrine-active, it is important to determine whether the amounts present in rodent diets are sufficient to affect sexual development. The present study consisted of in vitro and in vivo parts. In the in vitro portion, human hepatoma cells were transfected with either rat estrogen receptor (ER) alpha or beta plus an estrogen-responsive luciferase reporter gene. Genistein and daidzein were complete agonists at both ERs, genistein being more potent than daidzein, and both compounds were more potent at ER beta than ER alpha. In combined studies with estradiol, genistein exerted additive effects with estradiol in vitro. In the in vivo portion of the study, groups of six pregnant Sprague-Dawley females were fed one of the following four diets, and the pups were maintained on the same diets until puberty: (1) a natural-ingredient, open-formula rodent diet (NIH-07) containing 16 mg genistein and 14 mg daidzein per 100 g of feed; (2) a soy- and alfalfa-free diet (SAFD) in which casein and corn oil were substituted for soy and alfalfa meal and soy oil, respectively, that contained no detectable isoflavones; (3) SAFD containing 0.02% genistein (GE.02); or (4) SAFD containing 0.1% genistein (GE.1). In the GE.1 group, effects of dietary genistein included a decreased rate of body-weight gain, a markedly increased (2.3-fold) uterine/body weight (U/BW) ratio on postnatal day (pnd) 21, a significant acceleration of puberty among females, and a marginal decrease in the ventral prostate weight on postnatal day (pnd) 56. However, developmental differences among the groups fed SAFD, GE.02, or NIH-07 were small and suggested minimal effects of phytoestrogens at normal dietary levels. In particular, on pnd 21, the U/BW ratio of the GE.02 and NIH-07 groups did not differ significantly from that of the SAFD group. Only one statistically significant difference was detected between groups fed SAFD and NIH-07: the anogenital distance (AGD) of female neonates on pnd 1 whose dams were fed NIH-07 was 12% larger than that of neonates whose dams were fed SAFD. The results suggest that normal amounts of phytoestrogens in natural-ingredient rodent diets may affect one developmental parameter, the female AGD, and that higher doses can affect several other parameters in both males and females. Based on these findings, we do not suggest replacing soy- and alfalfa-based rodent diets with phytoestrogen-free diets in most developmental toxicology studies. However, phytoestrogen-free diets are recommended for endocrine toxicology studies at low doses, to determine whether interactive effects may occur between dietary phytoestrogens and man-made chemicals.     

Retinoid-induced hemorrhaging and bone toxicity in rats fed diets deficient in vitamin K

The recent increase in the clinical use of synthetic vitamin A compounds has led to concern of possible side effects. Some of these effects are known to be influenced by dietary levels of vitamin K. We therefore compared the toxic effects of 13-cis-retinoic acid (13cisRA), retinyl acetate (ROAc), and N-(4-hydroxyphenyl)retinamide (4HPR) in male Sprague-Dawley rats maintained on diets containing different levels of vitamin K. Animals were fed either an NIH-07 diet supplemented with menadione (3.1 ppm vitamin K3), an NIH-07 diet not supplemented with menadione, or an AIN-076 purified diet devoid of vitamin K. The retinoids had no effect on prothrombin times of animals fed the supplemented diet. When menadione was omitted from the diet, however, 4HPR-dosed animals had elevated prothrombin times. This effect was observed as early as Day 7 and was accompanied by one confirmed hemorrhagic death. 13cisRA-dosed animals showed no change in prothrombin times. In the high-dose ROAc group, there was a twofold increase in prothrombin times but only after prolonged dosing. In animals fed the NIH-07 diets, 13cisRA and ROAc induced multiple bone fractures at all dose levels. In contrast, 4HPR administered at the highest dose induced only one fracture in one animal. Animals fed the purified diet lost weight faster and diet sooner than those maintained on the other diets. Bone fractures were not observed in these animals because of early deaths resulting from hemorrhaging. For all retinoid-dosed groups maintained on the purified diet, changes in prothrombin times occured as early as 1 week. The order of effect was 4HPR greater than ROAc greater than 13cisRA, with increases in prothrombin times correlating with increases in hemorrhagic deaths. Hence, the degree of retinoid-induced hemorrhage, but not the incidence of bone fractures, was inversely related to vitamin K levels in the diet. 13cisRA and ROAc, but not 4HPR, caused a dose-dependent reduction in plasma osteocalcin, an effect that correlated with retinoid-induced bone effects. In contrast, serum alkaline phosphatase was elevated in animals dosed with 13cisRA or 4HPR but not in those dose with ROAc. For this enzyme, the electrophoretic pattern on agarose gel showed a decrease, compared to controls, in the major isozyme in serum of ROAc-dosed animals. Hence, plasma osteocalcin is a better predictor of retinoid-induced bone effects than serum alkaline phosphatase.     

Effects of pectin-containing diets on the hepatic macromolecular covalent binding of 2,6-dinitro-[3H]toluene in Fischer-344 rats

The influence of diets varying in pectin content on intestinal microfloral metabolic capacity of rats has been investigated as a possible mechanism for the alteration of toxicity of 2,6-dinitrotoluene (2,6-DNT) produced by these diets. Male F-344 rats were fed a purified diet (AIN-76A), AIN-76A plus 5% or 10% citrus pectin, or either of two cereal-based diets that vary in pectin content, NIH-07 or Purina Chow 5002. After 28 days, rats were given tritium-labeled 2,6-DNT (10 or 75 mg/kg po) and killed 12 hr later. Total hepatic macromolecular covalent binding (CVB) was determined by exhaustive extraction. The CVB of 2,6-DNT was found to be independent of diet at 10 mg/kg. However, at 75 mg/kg CVB was increased 40% by feeding 5% pectin in the purified diet and 90% by feeding 10% pectin in the purified diet. Animals fed Purina 5002 and NIH-07 had 135 and 150% higher CVB, respectively, than animals fed the purified diet alone and significantly greater CVB than animals fed the pectin supplemented diets. Elevated (two- to threefold) β-glucuronidase and nitroreductase activities, microfloral enzymes proposed to be involved in the activation of 2,6-DNT to a toxicant, were found in the cecal contents of animals fed the pectincontaining diets which correlated with a two- to threefold increase in total number of cecal anaerobes. These results suggest that pectin-induced changes in microflora may enhance hepatoxicity after high doses of 2,6-DNT.     

Effects of purified (AIN-76A) and natural-ingredient (NIH-07) diets on responses of BALB/c and B6C3F1 female mice to estradiol

Female BALB/c and B6C3F1 mice were examined after a 3-wk exposure to dietary estradiol (0, 400, 800, 1600, and 3200 ppb) in a purified (AIN-76A) or a natural-ingredient (NIH-07) diet. The use of AIN-76A was associated with a 9-13% greater (p less than 0.001) body weight and a 36-43% higher (p less than 0.001) serum cholesterol in both mouse genotypes when compared to mice fed NIH-07. Conversely, when fed NIH-07, both mouse genotypes had a 20-22% higher (p less than 0.003) serum urea nitogren and 2-3.5% higher erythrocyte count (p less than 0.001) and hemoglobin concentration (p less than 0.04) than when fed AIN-76A. Reduced erythrocyte parameters suggest that chronic feeding of the purified diet might result in anemia. No significant compound or diet-related differences were noted for serum creatinine, alanine aminotransferase, aspartate aminotransferase, or gamma-glutamyl transferase. Although there was no diet effect on absolute or differential white blood cell count, estradiol caused a decrease in the total white blood cell count (p less than 0.014) and an increase in the percentage of polymorphonuclear leukocytes (p less than 0.014) in BALB/c and decreased the percentage of lymphocytes (p less than 0.005) in B6C3F1 females. In addition, estradiol increased uterine weight and inhibited thymic and splenic weights in one or both genotypes. Spleen and thymus weight responses to estradiol were not significantly influenced by diet. However, the uterine weight responses to estradiol were apparently influenced by diet in both genotypes. In B6C3F1 mice, the uterus weighed more at each level of estradiol when mice were fed AIN-76A compared to NIH-07 diet. In BALB/c mice, this was true only at the two lower dietary concentrations of estradiol. In conclusion, mice fed the purified diet, AIN-76A, differed from those fed the cereal-based diet, NIH-07, in hematology, clinical chemistry, and uterine weight response to estradiol.     

Comparison of endpoints relevant to toxicity assessments in 3 generations of CD-1 mice fed irradiated natural and purified ingredient diets with varying soy protein and isoflavone contents

Diet is an important variable in toxicology. There are mixed reports on the impact of soy components on energy utilization, fat deposition, and reproductive parameters. Three generations of CD-1 mice were fed irradiated natural ingredient diets with varying levels of soy (NIH-41, 5K96, or 5008/5001), purified irradiated AIN-93 diet, or the AIN-93 formulation modified with ethanol-washed soy protein concentrate (SPC) or SPC with isoflavones (SPC-IF). NIH-41 was the control for pairwise comparisons. Minimal differences were observed among natural ingredient diet groups. F₀ males fed AIN-93, SPC, and SPC-IF diets had elevated glucose levels and lower insulin levels compared with the NIH-41 group. In both sexes of the F₁ and F₂ generations, the SPC and SPC-IF groups had lower body weight gains than the NIH-41 controls and the AIN-93 group had an increased percent body fat at postnatal day 21. AIN-93 F₁ pups had higher baseline glucose than NIH-41 controls, but diet did not significantly affect breeding performance or responses to glucose or uterotrophic challenges. Reduced testes weight and sperm in the AIN-93 group may be related to low thiamine levels. Our observations underline the importance of careful selection, manufacturing procedures, and nutritional characterization of diets used in toxicological studies.     

Adverse effect of a sucrose-based semipurified diet on development and postnatal growth of Fischer rats

A commercially prepared semipurified diet (Bio-Serve No. 0006Q) composed of chemically defined ingredients was fed to male and female F344 rats before mating, to females during gestation, and to the offspring after weaning. Compared to rats treated similarly but fed a standard, cereal-based chow (Agway CR RMH 3000), offspring on the semipurified diet exhibited markedly retarded growth. At 12 weeks of age, the deficits in body weight were accompanied by decreased absolute organ weights, increased organ-to-body weight ratios, increased serum cholesterol and triglyceride levels, and development of hepatic periportal lipidosis in both male and female offspring on the semipurified diet. The deficits in body weight are explained, in part, by decreased intake of the semipurified diet, most likely attributable to its high sucrose content. Investigators are cautioned about the inadequacy of certain semipurified diets for development and postnatal growth of F344 rats.     

Toxicity and carcinogenicity studies of phenylephrine hydrochloride in F344/N rats and B6C3F1 mice

Phenylephrine HCl was incorporated into feed given to male and female F344/N rats and B6C3F1 mice in studies of 14 days, 12 weeks, and 2 years duration. In 12-week studies, body weight gains decreased with dose, and deaths of male rats and mice occurred at concentrations of 5,000 ppm and above; however, no organ-specific toxicity was evident. During 2-year studies, body weights of rats receiving diets at 620 and 1,250 ppm and mice at 1,250 and 2,500 ppm ranged up to 16% less than control. Survival of high dose male rats was substantially greater than controls. Survivals of other dose groups of rats and mice were similar to controls. Chronic focal inflammation of the liver, and inflammation of the prostate were increased in dosed rats. No increases in neoplasms were observed in rats or mice consuming diets containing phenylephrine HCl for 2 years. The incidences of mononuclear cell leukemia and pheochromocytomas of the adrenal gland were decreased in dosed male rats. Approximate time weighted average doses ranged up to 54 mg/kg/day for rats and 280 mg/kg/day for mice during the 2-year studies.     

Three-generation evaluation of dietary para-nonylphenol in CD (Sprague-Dawley) rats.

This study evaluated the potential for dietary para-nonylphenol (NP; CAS No. 84852-15-3) to affect parental fertility and growth and development of three offspring generations in CD (Sprague-Dawley [SD]) rats, including sperm counts across generations to determine the validity of equivocal reductions observed in the F2 generation by R. E. Chapin et al. (1999, Toxicol. Sci. 52, 80-91). Male rat kidney toxicity was also examined based on inconsistent observations in NP-exposed rats at 2000 ppm but not at 200 or 650 ppm in Purina 5002 (H. C. Cunny et al., 1997, Regul. Toxicol. Pharmacol. 26, 172-178) and at all of these NP concentrations in NIH-07 diet (R. E. Chapin et al., 1999, Toxicol. Sci. 52, 80-91). Concentrations were 0, 20, 200, 650, and 2000 ppm NP in Purina 5002 diet and 0 and 650 ppm NP in NIH-07 diet. 17beta-estradiol (E2) was used as a positive control at 2.5 ppm in Purina 5002 diet. There were no NP effects on any reproductive parameters in any generation, including sperm counts. Kidney toxicity (histopathology) occurred at 650 and 2000 ppm with no clear difference for the two diets. Ovarian weight was decreased at 2000 ppm NP in all generations, with no effect on reproduction. Dietary E2 at 2.5 ppm caused renal, reproductive, and developmental (lactational and peripubertal) toxicity in all generations. This study confirmed that dietary NP is not a selective reproductive toxicant with an no observable adverse effect level (NOAEL) of > 2000 ppm ( approximately > 150 mg/kg/day) and provided an NOAEL for male rat kidney toxicity of 200 ppm NP (approximately 15 mg/kg/day).     

Rat and poultry feeding studies with soybean meal produced from imidazolinone-tolerant (CV127) soybeans

The safety and nutritional properties of CV127 soybeans were evaluated in rat and broiler feeding studies. Some episodic differences were observed between rats fed CV127, Conquista, and the standard diet for the endpoints examined. None of these differences were considered treatment related, adverse, or biologically meaningful. In general, birds fed diets containing CV127, Conquista, or Monsoy 8001 showed no significant differences in growth and performance response variables. Chickens fed diets containing Coodetec 217 had lower body weight and weight gain for all developmental periods compared to CV127, but no significant differences were found in feed conversion for the two diets during any development period. The results of both feeding studies demonstrate that CV127 soybeans are as safe, wholesome, and nutritionally valuable as the other soybean meals tested, including those varieties for which histories of safe use have been established and well documented.     

Toxicology and carcinogenesis studies of pentachlorophenol in rats.

Pentachlorophenol (PCP) has been used as an herbicide, algaecide, defoliant, wood preservative, germicide, fungicide, and molluscicide. A 28-day toxicity study of PCP in F344/N rats of both sexes was conducted to select dose levels for a carcinogenicity study. Groups of 10 male and 10 female rats were given 0, 200, 400, 800, 1600, or 3200 ppm PCP in feed for 28 days. The incidences of minimal to mild hepatocyte degeneration in males and females exposed to 400 ppm or greater and the incidences of centrilobular hepatocyte hypertrophy in the 3200-ppm groups were increased. For carcinogenicity studies, groups of 50 male and 50 female F344/N rats were fed diets containing 200, 400, or 600 PCP for 2 years. A stop-exposure group of 60 male and 60 female rats received 1000 ppm of PCP in feed for 52 weeks and control feed thereafter for the remainder of the 2-year studies; 10 male and 10 female rats were evaluated at 7 months. Survival of 600-ppm males was significantly greater than that of the controls; survival of all other exposed groups was similar to that of the control groups. Mean body weights of the 400- and 600-ppm groups were generally less than those of the controls throughout the studies. There was no evidence of carcinogenic activity of PCP in male or female rats fed diets containing 200, 400, or 600 ppm for 2 years. Stop-exposure study males and females regained a transitory body weight reduction by the end of the 2 year study, and males had better survival than the controls. At a 7-month interim evaluation, the incidences of centrilobular hypertrophy in stop-exposure males and females exceeded those in the controls. At 2 years, malignant mesothelioma originating from the tunica vaginalis was present in 9 1000-ppm males and 1 control male (p = 0.014). Nasal squamous cell carcinomas were present in five 1000-ppm males and 1 control male. This incidence was not significantly increased but exceeded the historical control range (0-4%). Based on the increased incidences of mesotheliomas and nasal tumors, there was some evidence of carcinogenic activity of PCP in male rats given a diet containing 1000 ppm for 1 year followed by control diet for 1 year. There was no evidence of PCP carcinogenic activity in stop-exposure female rats.     

Evaluation of 90-day oral rat toxicity studies on the food additive,gum ghatti

Gum ghatti, a polysaccharide of natural origin, is used in foods as a thickening, gelling, emulsifying and stabilizing agent. In a 90-day toxicity study following Organization for Economic Co-operation and Development (OECD) Guideline #408, male and female Sprague–Dawley rats were exposed to 0 (control), 0.5, 1.5 and 5% gum ghatti in AIN-93M basal diet. Expected changes included increased full and empty cecal weights in 5% groups. Incidentally 2/10 females from the 5% gum ghatti group had a single colon ulcer with associated acute inflammation. In a second 90-day study increased cecal weights were present in Sprague–Dawley females exposed to 5% gum ghatti in AIN-93M and NIH-07 basal diets. A single colon ulcer with associated acute inflammation occurred in 1/20 control females given AIN-93M basal diet. The colon ulcers were considered a sporadic change possibly attributable to AIN-93M basal diet. In the second study a few statistically significant alterations in clinical chemistry were considered sporadic and unrelated to treatment. Feed consumption among treated and control groups was similar for each sex. Gum ghatti intake at the 5% dietary level ranged from 3044 to 3825 mg/kg body weight/day. The 5% dietary administration was a NOAEL in both studies. NOAELs for males and females in the first study were 3044 and 3309 mg/kg/day, respectively. NOAELs for females in the second study were 3670 and 3825 mg/kg/day for AIN-93M and NIH-07 diets, respectively.     

Chronic effects of agar,guar gum,gum arabic,locust-bean gum,or tara gum in F344 rats and B6C3F1 mice

Diets containing 25,000 (2.5%) or 50,000 ppm (5.0%) agar, guar gum, gum arabic, locust-bean gum or tara gum were fed to groups of 50 male and 50 female F344 rats and B6C3F₁ mice for 103 wk. Separate groups of 50 rats and 50 mice of each sex served as controls for each study. There were no significant differences in survival between any of the dosed groups of rats or mice and their respective control groups. Depressions in body-weight gain greater than 10% for dosed groups relative to their respective control groups were observed for male (low dose only) and female mice fed diets containing agar, female mice fed diets containing guar gum (high dose only), male mice fed diets containing locust-bean gum (high dose only) and male and female mice fed diets containing tara gum (high dose only). Depressions in body-weight gain greater than 5% were observed for female rats fed diets containing agar, guar gum or gum arabic. There were no histopathological effects associated with the administration of the test materials. Under the conditions of these bioassays, none of the five polysaccharides was carcinogenic for F344 rats or B6C3F₁ mice of either sex.