title: Effect of Dietary Restriction on Toxicology and Carcinogenesis Studies in F344/N Rats and B6C3F1 Mice.
abstract: Studies were conducted to compare outcomes when four chemicals were evaluated under typical NTP bioassay conditions as well as under protocols employing dietary restriction. Specific experiments were designed to evaluate the effect of diet restriction on the sensitivity of the bioassay toward chemical-induced chronic toxicity and carcinogenicity and to evaluate the effect of weight-matched control groups on the sensitivity of the bioassays. Two chemicals, butyl benzyl phthalate and t-butylhydroquinone, were administered in feed; one chemical, salicylazosulfapyridine, was administered in corn oil by gavage; and one chemical, scopolamine hydrobromide trihydrate, was administered in distilled water by gavage. In each of four protocols, the effects of the chemical were assessed by a comparison between a group exposed to a single dose concentration of the study chemical and a nonexposed control group. F344/N rats and B6C3F1 mice were fed NIH-07 diet either ad libitum or in amounts that restricted mean body weights according to the following design requirements. For the core bioassay, groups of 50 to 60 ad libitum-fed animals were allotted to a control group and three dosed groups for approximately 104 weeks or up to 128 weeks (t-butylhydroquinone study). The comparison between the control group and the group receiving the highest dose was used to represent the outcome of the bioassay under ad libitum feeding protocols. In a second comparison, outcomes from the group receiving the highest dose were compared with a weight-matched group of 50 to 60 untreated controls; the weight-matched controls received feed in amounts restricted so that the mean body weight matched the mean body weight of the dosed group. Two additional groups of 48 to 60 animals (one control and one dosed group) were offered feed in amounts that limited the mean body weight of the control group to approximately 85% that of the controls fed ad libitum under the first protocol. Animals assigned to this dietary restriction paradigm were evaluated after 104 weeks or 130 weeks (t-butylhydroquinone). A fourth protocol was em- loyed to evaluate whether an additional period of exposure (up to 1 year) would influence the neoplasm profile of animals fed a restricted diet. Two groups of approximately 50 animals (one control and one dosed group) in the butyl benzyl phthalate, salicylazosulfapyridine, and scopolamine hydrobromide trihydrate studies received restricted diets, as under the third protocol, for 3 years or until survival in either group was reduced to 20%. Butyl benzyl phthalate caused an increased incidence of pancreatic acinar cell neoplasms in ad libitum-fed male rats relative to ad libitum-fed and weight-m atched controls. This change did not occur in rats in the restricted feed protocol after 2 years; however, acinar cell adenomas were observed in three exposed, feed-restricted males at 30 months. Feed restriction is known to influence the incidence of pancreatic acinar cell neoplasms and may have prevented the full expression of this chemical-induced effect. Butyl benzyl phthalate also caused an increased incidence of urinary bladder neoplasms in female rats in the 32-month restricted feed protocol. The incidences of urinary bladder neoplasms were not significantly increased in female rats in any of the 2-year protocols, suggesting that the length of study, and not body weight, was the primary factor in the detection of this carcinogenic response. Salicylazosulfapyridine caused an increased incidence of urinary bladder papillomas in male rats fed ad libitum relative to ad libitum-fed and weight- matched controls. This increase was associated with an increased incidence of urinary bladder calculi; the incidences of urinary bladder concretions, dilatation, and hyperplasia were also increased in dosed males. The incidences of urinary bladder papillomas and calculi were not increased in male rats receiving salicylazosulfapyridine that were fed restricted diets. In male mice, salicylazosulfapyridine caused an increased incidence of liver neoplasms relative tsms relative to the ad libitum-fed and weight-matched controls. This increase did not occur in the restricted feed protocols. Liver neoplasms in mice are greatly influenced by body weight, and the marked mean body weight reduction observed in dosed male mice in the restricted feed protocols may have overridden the carcinogenic response. Neither t-butylhydroquinone nor scopolamine hydro bromide trihydrate caused increased neoplasm incidences under any of the experimental protocols. Results consistently show that feed restriction caused decreased incidences of neoplasms and nonneoplastic lesions at a variety of anatomic sites in control and dosed animals. Furthermore, the sensitivity of the bioassay to detect a carcinogenic response was altered by dietary restriction: two of the four chemicals caused increased incidences of neoplasms at three sites when evaluated under a standard ad libitum feeding protocol for 104 weeks. When control and dosed groups were subjected to dietary restriction, none of these three sites was detected as a target of carcinogenesis after 2 to 3 years. Rather, one different site of carcinogenesis was detected after 32 months. When dosed animals in the ad libitum feeding protocol were compared to weight-matched control groups, three sites were identified as targets of carcinogenesis and corresponded to the three sites discovered under the ad libitum feeding protocol. The magnitude of the response was greater when the weight-matched controls protocol was used. Dietary restriction of dosed and control animals decreased the sensitivity of these carcinogenesis bioassays. Regarding the future use of dietary restriction regimens in long-term studies, only limited conclusions can be drawn because only four chemicals were evaluated and none of these proved to be a strong carcinogen. However, the results of these studies are consistent with previous findings that dietary restriction increases survival rates and decreases the incidences of neoplasms and nonneoplastic lesions at a variety of sites in rats and mice. This association between reduced body weights and decreased neoplasm incidences underlines the necessity that the doses selected for chronic studies not exceed "minimally toxic doses" so that no marked body weight reductions (or increases) will occur in the dosed groups. Such body weight changes complicate the detection of carcinogenic effects. The following tables summarize and compare the findings from ad libitum-fed, weight-matched, and feed-restricted groups for each chemical. Tabular Summary of Dietary Restriction Study of Butyl Benzyl Phthalate is available in web version of this document. TabularSummary of the Dietary Restriction Study of t-Butylhydroquinone is available in web version of this document. TabularSummary of the Dietary Restriction Studies of Salicylazosulfapyridine is available in web version of this document. TabularSummary of the Dietary Restriction Study of Scopolamine Hydrobromide Trihydrate is available in web version of this document.