Variability of selected nutrients and contaminants monitored in rodent diets: a 6-year study.

The results are given from monitoring a commercial closed-formula cereal-based rodent diet (Purina 5010), two open-formula cereal-based diets (NIH-31 and NIH-07), and one purified diet (AIN-76) for selected nutrients and contaminants. The observed concentrations of nutrients (protein, fat, vitamin A, and thiamine) approximated the manufacturer specifications for closed-formula cereal diet, while the average concentrations of nutrients found in the open-formula cereal diets were well above the nominal concentrations. Nominal concentrations for these open-formula diets tended to be close to the minimum values that were observed. Except for protein levels, greater variation in nutrient concentrations was found in the purified diet than in the cereal diets, but the variation of contaminants was about equal in the two types of diets. Open- and closed-formula cereal diets appear to be very similar to each other in the degree of variation of nutrients and contaminants. Cadmium, lead, and selenium are the constituents of greatest concern in assuring the quality of the rodent diets that were evaluated.

Quality changes in animal feed resulting from extended storage.

The objective of this study was to determine if extended storage of animal feed after autoclaving had an effect on the quality of feed which could be deleterious to experimental animals. Fresh animal feed was autoclaved each week and placed in an environmentally controlled storage room (18.5 +/- 2 degrees C with 50 +/- 5% RH). This process provided feed on a weekly basis that had a consistent storage time of 10, 74 or 130 days. Weanling male and female BALB/c and B6C3F1 mice were the test animals. The animals were housed four to a cage and were started on the experiment in two replicates separated by one week. The study was terminated after 13 weeks. Food consumption and body weights were measured weekly and serum protein levels analyzed upon termination of the study. Statistical analysis of the results showed several interactions among gender, replication and mouse genome, but no significant differences were present among treatment groups for the serum proteins or weight ratios. The animals appeared to be normal, and a histological examination of the liver showed no signs of disease or nutritional disorders. This study indicates that feed can be stored up to 130 days after autoclaving without apparent deleterious effects on animals receiving the feed.

A rapid procedure to efficiently clean blenders repeatedly used to prepare dosed feed for bioassays.

An effective and economical procedure is described to clean blending equipment repeatedly used to prepare dosed feed for animal bioassays. The cleanup procedure was evaluated with 2 types of blenders against 10 test chemicals with a broad spectrum of polarities by separately spiking the agents into feed at various levels. Analytical data illustrating the level of contamination at each interval of the multi-step cleanup procedure are presented for each test chemical. Analytical chemical methods and efficiencies of the cleaning steps with the feed admixtures are discussed. The procedure was tested and adopted for use at the National Center for Toxicological Research (NCTR).

Benzidine-congener-based azo dyes: assays for purity and residues in feces from dosed rats.

Analytical methods were required to determine purities of benzidine-congener-based azo dyes and residues of the intact dyes in feces from rats before valid metabolism studies of such compounds could be conducted. A procedure is described for purity assays based on reduction of the dyes with stannous chloride followed by gas chromatography of the released free amine. Sixteen different samples of commercial dyes based on three benzidine congeners were assayed; purities ranged from 26.4 to 83.4%. Several dyes were also shown to be partially purified by cold water washes. A method to determine two intact dyes in feces from dosed rats, which consisted of extraction with dimethylformamide: water, clean-up by a rapid procedure using an octadecylsilane column, and quantification by ion-pair high-pressure liquid chromatography (HPLC) is reported. minimum detectable levels of both dyes in feces are 0.2 ppm. Excretion profiles based on parallel HPLC and radioassays of feces from rats dosed with 14C-labeled Direct Blue 15 and Direct Red 2 are presented. Based on radioassays, about 74% of each dose was excreted via the feces; however, HPLC assays showed that only about 11% of each dose was present as intact dye in the excrement.

Metabolism and distribution of two 14C-benzidine-congener-based dyes in rats as determined by GC, HPLC, and radioassays.

Absorption, metabolism and tissue distribution studies were conducted in the rat with 14C-biphenyl ring-labeled Direct Blue 15, a 3,3'-dimethoxybenzidine (DiMxBzd) based azo dye; Direct Red 2, based on 3,3'-dimethylbenzidine (DiMeBzd) and corresponding benzidine congener amines. Single oral doses of the 14C-labeled dyes (12 mg/kg, 62 microCi/kg) and molar equivalent doses of the respective amines were administered and urine and fecal samples collected at intervals up to 192 hours. Urine specimens were analyzed for 14C content and further characterized by EC/GC for free amines, acetylated metabolites, and conjugates. Feces were assayed for 14C content and for unchanged dosed dyes or amines by HPLC. A comparison of the metabolism of Direct Blue 15 with its base DiMxBzd, indicated that the base was more extensively metabolized and that most of the 14C in various extracts was identified as known metabolites. The metabolism of Direct Red 2 compared with its base, DiMeBzd, indicated that the base was more extensively metabolized, yet only a small percentage of the 14C in extracts was identified as known metabolites. Most of the 14C present in the urine could not be extracted with benzene nor chloroform, indicating high polarity. Distribution studies conducted with both dyes showed that liver, kidney, and lung accumulated and retained higher levels of 14C than other tissues (at 72 hrs). Peak levels of 14C, which occurred 8-12 hours after dosing, were significantly higher with Direct Red 2 than Direct Blue 15. Tissue distribution data (72 hr) for rats dosed with the free amines compared with the dyes showed a generally lower but similar distribution pattern.

Determination of tibric acid and clofibrate in animal feed, wastewater and hunan urine.

Analytical chemical procedures are described to determine residues of the drugs clofibrate and tibric acid in animal feed, wastewater, and human urine. Clofibrate was extracted from animal feed and human urine with hexane, whereas residues from wastewater were collected on a Sep-PakTM then eluted with methanol for analysis. Clofibrate residues from the feed, wastewater, and urine were analyzed by high-pressure liquid chromatography (HPLC) with minimum detectable levels (MDL) of about 40, 0.5 and 1.0 ppb, respectively. Tibric acid was extracted from animal feed with 90% methanol and 10% 0.1 N NaOH, whereas wastewater and human urine were acidified with 12 N HCl and then extracted with benzene. The MDL for tibric acid in feed by electron capture/gas chromatography (EC/GC) and HPLC were about 40 ppb and 2.0 ppm, respectively. Residues from these extracts that contained more than 5 ppm of tibric acid were analyzed by HPLC, whereas GC was required for levels below 5 ppm. The GC procedures, which required that tibric acid be derivatized (methylated) prior to analysis, had MDL of 0.1 and 1.0 ppb for wastewater and human urine, respectively. Data are also presented concerning partition values, stability of the compounds in animal feed, and recoveries of the compounds from the three substrates.

Commercial laboratory animal diets: toxicant and nutrient variability.

A commercial rodent feed was analyzed for a series of nutrients and potential contaminants during a 5-yr period. Annual average Cu and vitamin A concentrations were generally at least 12% lower than the approximate concentrations listed by the manufacturer, whereas Ca, protein, and vitamin B1 were within +/- 5% and fat and Zn within +/- 8% of the manufacturer's specifications. Frequently, Se was found at concentrations at which it has been shown to interact with the process of chemical carcinogenesis. DDT, dieldrin, Cd, and Pb were occasionally close to concentrations known to have biological effects.

A toxicological risk assessment procedure: a proposal for a surveillance index for hazardous chemicals.

Health-oriented government agencies responsible for the protection of the public from possible adverse effects, such as that posed by chemical residues in the food supply, must establish priorities on regulatory action as well as for manpower to conduct monitoring programs. A new procedure entitled Surveillance Index (SI) is presented as a technique to evaluate toxic materials that are potential candidates as residues in food supplies and to assign an index number that identifies a relative hazard to the public among the various residues. The SI numbers can be used as criteria by which chemical residues should be monitored whenever conditions such as time and available resources are limiting factors.

Chemical surveillance and quality assurance for preparation of dosed (2-AAF) animal feed (ED01 study).

Protocol development of the ED01 experiment included assurances that only minimal levels of contaminants were present and correct concentrations of 2-AAF were present in animal feed. Laboratory and administrative controls were developed to assure proper feed packaging and delivery, and to conduct a personnel surveillance as well as a chemical surveillance of work environs to ensure safe work areas. Animal feed and ancillary animal supplies were monitored prior to use to provide assurances that acceptable levels of nutrients were present and to prohibit the entrance of unacceptable levels of contaminants such as pesticides and heavy metals.

Trace analysis of 2,4,5-trichlorophenoxyacetic acid, its glycineamide, and their alkaline hydrolyzable conjugates in mouse blood, urine, and feces.

Chemical methods were developed for the trace analysis of the herbicide 2,4,5-trichlorophenoxyacetic acid, its glycineamide, and their alkaline hydrolyzable conjugates in mouse blood, urine, and feces. The salient elements of the methods are extraction of the free acids with benzene, methylation, cleanup on a silica gel column, and quantification via electron-capture GLC. Any unextracted conjugates remaining in the substrates are then subjected to alklaline hydrolysis, and the liberated 2,4,5-trichlorophenoxyacetic acid is assayed. Data are presented concerning recoveries of the compounds from the three spiked substrates. The utility of the procedures is illustrated by a preliminary pharmacolinetic study employing parallel electron-capture GLC and radioassays of the three substrates from mice injected with a single intravenous dose of 14C-2,4,5-trichlorophenoxyacetic acid. GLC characteristics and partition values of the the compounds and hydrolysis of the glycineamide under various conditions also are discussed.

Analysis of chlorinated insecticides and congeners.

A review on the state-of-the-art for analysis of chlorinated insecticides and their congeners is presented. Tables are included that show comparisons among gas chromatographic systems, high speed liquid chromatographic systems and sample preparation methods as well as an overview on sample cleanup.