560683005 111 1984 NEPIS online hardcopy LM 20120301 single page tiff ORNL Oak Ridge National Laboratory Operated by Martin Marietta Energy Systems, Inc. for the Department of Energy Oak Ridge, Tennessee 37831 ORNL-6035 United States Environmental Protection Agency Office of Toxic Substances Washington, DC 20460 EPA-560/6-83-005 May 1984 Toxic Substances Nutritional Requirements and Contaminant Analysis of Laboratory Animal Feeds image: ------- Printed in the United States of America. Available from National Technical Information Service U.S. Department of Commerce 5285 Port Royal Road, Springfield, Virginia 22161 NTIS price codes—Printed CopyA06 Microfiche A01 This report was prepared as an account of work sponsored by an agency of the United States Government. Neither theUnitedStatesGovernment nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency" thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. image: ------- ORNL-6035 EPA 560/6-83-005 May 1984 NUTRITIONAL REQUIREMENTS AND CONTAMINANT ANALYSIS OF LABORATORY ANIMAL FEEDS by Bimal C. Pal Robert H. Ross Chemical Effects Information Center Information Division Oak Ridge National Laboratory Oak Ridge. TN 37831 and Harry A. Milman Health and Environmental Review Division Office of Tozic Substances U.S. Environmental Protection Agency Washington, DC 20460 Interagency Agreement DW930141-01-1 Date Published - May 1984 Prepared For OFFICE OF TOXIC SUBSTANCES U.S. ENVIRONMENTAL PROTECTION AGENCY WASHINGTON. DC 20460 Prepared by the OAK RIDGE NATIONAL LABORATORY Oak Ridge. Tennessee 37831 operated by MARTIN MARIETTA ENERGY SYSTEMS, INC. for the U.S. DEPARTMENT OF ENERGY under Contract No. DE-AC05-840R21400 image: ------- This report has been reviewed by the Office of Toxic Substances, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recom- mendation for use. ii image: ------- CONTENTS Page LIST OF FIGURES v LIST OF TABLES vii ACKNOWLEDGMENTS iz EXECUTIVE SUMMARY zi 1 INTRODUCTION 1 2 NUTRITIONAL REQUIREMENTS FOR LABORATORY ANIMALS - COMPARISON OF THE DATA PROPOSED IN THE EPA GOOD LABORATORY PRACTICE STANDARDS WITH THOSE OF COMMERCIAL PROCEDURES, NIH, AND AIN 5 3 EVALUATION OF DIFFERENT TYPES AND FORMS OF DIETS 6 3.1 Types of Diets - Advantages and Disadvantages 6 3.1.1 Natural-Ingredient Diets 6 3.1.2 Purified Diets 6 3.1.3 Chemically Defined Diets 6 3.1.4 Open Formula Diets 12 3.1.5 Closed Formula Diets 12 3.1.6 Quasi-Open Formula Diets 12 3.2 Forms of Diets 12 3.3 Autoclaving of Diets 14 3.4 Discussion 15 4 DIETARY INTAKE 17 5 VITAMINS AND MINERALS USED IN ANIMAL DIET FORMULATIONS 18 5.1 Vitamins 18 5.2 Minerals 18 6 INGREDIENTS USED IN LABORATORY ANIMAL DIETS 22 7 CONTAMINANTS AND PRESERVATIVES IN ANIMAL FEEDS 24 7.1 Introduction 24 7.2 Biological Contaminants 24 7.2.1 Estrogens 24 7.2.2 Aflatozins 26 7.2.3 Tricothecene Tozins 27 7.3 Nitrosamines 27 7.4 Preservatives 30 7.5 Tolerance Limits of Contaminants and Preservatives in Animal Feed 31 7.5.1* Considerations, for Establishing Tolerances for Contaminants 36 7.5.2 Suggested Changes in the Tolerance Limits for the Contaminants Listed in the EPA GLP 36 iii image: ------- 7.5.3 Inconsistencies in EPA and FDA Tolerance Limits for Contaminants 44 8 MONITORING AND ANALYSIS OF CONTAMINANTS IN FEED 45 9 INTERACTION OF DIETARY COMPONENTS 47 9.1 Interplay Among Zn, Se, As, Vitamins, Amino Acids, Fatty Acids, and Phytate 47 9.2 Ca:P Ratio 47 9.3 K:Na Ratio 47 9.4 Enzyme Induction and Inhibition of Neoplasia by Minor Dietary Constituents, Feed Additives, and Contaminants 48 9.4.1 Enzyme Induction by Dietary Constituents 48 9.4.2 Enzyme Induction by Feed Additives 48 9.4.3 Enzyme Induction by Contaminants in Animal Feed ... 48 9.4.4 Inhibition of Neoplasia 49 9.4.5 Monitoring of Induced Enzyme Activity 49 9.4.6 Enzyme Induction and Genetic Differences 49 10 SPECIAL TOPICS 50 10.1 Phytates 50 10.2 Arsenic 50 10.3 Selenium 51 11 ANALYSIS OF PUBLIC COMMENTS ON THE PROPOSED EPA GUIDELINES FOR DIETARY REQUIREMENTS AND CONTAMINANT ANALYSIS 52 11.1 Criticisms of Proposed Guidelines and Related Discussion 52 11.2 Analysis of Specific Comments 53 11.2.1 Shelf Life Restrictions 53 11.2.2 Feed Meal 53 11.2.3 Calcium, Phosphorus, Iron, and Cobalt 53 11.2.4 Vitamins 54 11.2.5 Choline and Inositol 54 11.2.6 Alternative Sources of Minerals 55 11.2.7 Dicalcium Phosphate 55 11.2.8 Fat 55 11.2.9 Ash 55 11.2.10 Air Dry 56 11.2.11 Units of Measurement 56 LITERATURE CITED 57 Appendix A. National Institutes of Health Standard for Nutrient and Chemical Contaminant Analyses of Laboratory Animal Diets 71 Appendix B. Conversion Factors for Selected Vitamins 73 Appendix C. Letter from Edward J. Ballitch 74 Appendix D. Abbreviation List 75 Appendix E. Additional Literature References 76 iv image: ------- LIST OF FIGURES Figure Page 1 Growth curve for rabbit (New Zealand White) 8 2 Growth data for guinea pig (Ft. Detrick Dunkin Hartley) 9 3 Growth curves for fifteen breeds of dog 10 image: ------- LIST OF TABLES Table Page 1 Approximate Nutrient Composition of EPA, NIH, AIN and Commercial Laboratory Animal Diet Formulations 2 2 Estimated Nutritional Requirements of Laboratory Animals 3 3 Normal Growth. Rate of the Hamster, Rat, and Mouse 7 4 Reproduction and Life Span Data on Selected Laboratory Animals 11 5 Concentrations of Selected Nutrient and Toxic Components in Random Samples of Rodent Natural Product Diets 13 6 Vitamins, Minerals, and Animal Diet Formulations 19 7 Trace Minerals Included in the GRAS List 21 8 Ingredients Commonly Used in Laboratory Animal Diets 23 9 Estrogens in Animal Feed 25 10 Fusarium Toxins 28 11 Contaminants and Preservatives in Laboratory Animal Feeds 32 12 Pesticides and Contaminants in Laboratory Animal Feeds 33 13 Pesticides in Laboratory Animal Feeds 34 14 Normal Screening Levels of Detection 37 15 Biologically Effective Concentrations of Selected Organic Feed Contaminants 38 16 Biologically Effective Concentrations of Selected Heavy Metals 39 17 Interaction of Dietary Factors with Toxicants 40 18 Results of Analyses of Laboratory Animal Diets 43 19 References to the Methods of Analysis of Contaminants in Animal Feed and Relevant Statistical Data 46 vii image: ------- ACKNOWLEDGMENTS We have received assistance from many people in the preparation of this document. Dr. Gene Bingham, veterinarian. Biology Division, Oak Ridge National Laboratory, and Dr. Joseph J. Enapka, nutritionist. National Institutes of Health, have generously shared their expertise in this area. We are indebted to Betty J. Ward and Edward J. Ballitch of the Food and Drug Administration for enlightening us on the latest regulatory information. Dr. John C. Chah, Zeigler Bros., and Dr. Charles R. Adams, Hoffmann-La Roche, provided information on the stability of fats and vitamins. Thanks are due to Jon H. Ford, Manager, Ralston Purina Company Plant, Richmond, Indiana, for information on the current manufacturing practices in the laboratory animal feed industry. We have received invaluable advice, comments, and suggestions during the preparation of this document from Dr. Damon C. Shelton, Director, Laboratory of Special Chows Research Department, Ralston Purina Company, St. Louis, Missouri. Dr. Harry A. Milman of the U.S. Environmental Protection Agency provided us with a copy of the public comments on the USEPA Dietary Requirements under the Good Laboratory Practice Standards and general guidance during the preparation of this document. Thanks are due to Drs. Joseph J. Enapka (NIH), Kenneth C. Hayes (Harvard), Paul M. Newberne (MIT), and H. Leroy Shilt (Ralston Purina) for their in-depth reviews of this document. We greatly appreciate the assistance of Janice Pruett and Carolyn Seaborn for extensive literature searching and of Donna Stokes and Frances Lit- tleton for the very demanding task of typing. image: ------- EXECUTIVE SUMMARY The primary objectives of this report are to present information concerning the nutritional requirements of several commonly used labora- tory animal species (i.e., mouse, rat, hamster, guinea pig, rabbit, and dog) and to discuss various aspects of the problem of contamination of laboratory animal feeds. In addition, this document discusses the dif- ferent types of laboratory animal diets (e.g., open vs closed formula), the ingredients used in these diets, the interaction of dietary com- ponents, and the public comments received respective to the EPA proposed guidelines for the nutrient composition of laboratory animal diets. Much of the data are presented in tabular form as this method seemed the most appropriate. The principal focus of the discussion on nutritional requirements of laboratory animals is a comparison of the proposed EPA guidelines for rodent diets with the diet compositions for rodents of selected commer- cial laboratory animal feed manufacturers and with the recommendations of the National Academy of Sciences (NAS) regarding the nutrient compo- sition of laboratory animal diets. These comparisons revealed the fol- lowing: (1) commercial feed manufacturers are generally in good agree- ment with the dietary requirements proposed by EPA for rodents; and (2) the levels of nutrients proposed by EPA and found in the commercial feeds are usually higher than those recommended by the NAS. These higher levels result from nutrient concentrations in commercial diets being formulated to compensate for losses during the manufacture and storage as well as for differences in biological availability of nutrients. The different types of diets include natural-ingredient, purified, chemically defined, open formula, closed formula, and quasi—open for- mula. The merits and demerits of the open formula versus closed formula diet are a highly controversial issue. With respect to ingredients used in laboratory animal diets„ all of the ingredients listed in the proposed EPA guidelines are used in the National Institutes of Health (NIB) and in the selected commercial diet formulations reviewed. However, some ingredients are included in the NIH and commercial diet formulations that are not included as approved ingredients in the proposed EPA guidelines (DSEPA 1979). Contaminants in laboratory animal feeds arise from biological as well as nonbiological sources. Biological contaminants originate from microorganisms and are mainly estrogens, aflatozins, and trichothecene toxins. Nonbiological contaminants are usually pesticide residues, nitrosamines, and heavy metals. The EPA proposed guidelines list toler- ances for certain contaminants and preservatives, and commercial feed manufactures list maximum acceptable levels for contaminants. For some contaminants (e.g., lindane tfnd cadmium), the maximum allowable levels in commercially manufactured feeds exceed the EPA proposed tolerance levels. No preservatives are allowed according to the proposed EPA xi image: ------- guidelines whereas, for all the commercial producers whose data were reviewed, the preservative butylated hydroxyanisole is used in diets containing animal fats and the preservative ethoxyquin is used to prevent oxidation of vitamin A in diet formulations. Just as the presence of contaminants in the feed can affect the outcome of a long-term toxicity study, the interaction of dietary com- ponents can also play a determining role on the results of a chronic toxicity study. For example, selenium possesses anticarcinogenic pro- perties in animals and humans, and the uptake of selenium can be dimin- ished by zinc (Schrauzer 1977). Knowledge of such interactions would obviously be important for an investigator. Several commenters (comments regarding the proposed EPA guidelines) disagreed with the guidelines for nutrient composition of laboratory animal diets on the following grounds: (1) lack of feasibility; (2) practicality; (3) scientific justification; (4) lack of harmony with the Food and Drug Administration and the Department of Agriculture; (5) lack of incentive to continue research on diet development; and (6) failure to promote standardization of the diet. In conclusion, it should be noted that virtually no information exists with respect to the nutrient requirements of laboratory animals beyond the growth and reproductive segments of their life spans. There- fore, it is not known if the nutrient levels proposed by EPA and those in commercial diet formulations are the optimum for a lifetime bioassay study. Research in this area is obviously needed. In addition, the proposed EPA guidelines for diet composition are stated as the recommen- dations for rodents. However, some confusion arises since no dietary requirement is proposed for vitamin C which is needed in the diet of guinea pigs. Further clarification, or possibly diets recommended for individual species, would obviate this confusion. xii image: ------- 1. INTRODUCTION The coverage in this treatise has been restricted to six laboratory animal species: mouse, hamster, rat, guinea pig, rabbit, and dog. The purpose of this document is to provide the EPA with relevant facts and figures to assist in the revision of the Appendix B, Dietary Require- ments and Contaminant Analysis, of the Proposed Good Laboratory Practice Standards for Health Effects (USEPA 1979). Discussion and literature coverage of the nutrient requirements of different laboratory animals has been brief since this information is already available in the excel- lent monographs published by the National Academy of Sciences (NAS 1974, 1977, 1978). However, Appendix D contains a list of references not cited in this report but pertinent to the area of nutritional require- ments of laboratory animals; many of these are cited in the NAS publica- tions. The manufacture of the natural-ingredient diet, which is the most widely used animal diet, is quite involved and requires an expensive outlay. This is why almost all laboratories depend on the feed manufac- turer to provide an adequate and nutritious diet for the animals. Also, these commercial diets have long historical backgrounds and reliability. Representative data on the composition of animal feed from two of the more prominent producers have been collected and presented, along with data on diet composition from the National Institutes of Health (NIH) and the American Institute of Nutrition (AIN) (see Table 1), to provide the basis for comparison with the proposed EPA guidelines (also included in Table 1) and discussion of the state-of-the-art of dietary require- ments. Table 2 lists data on the nutritional requirements of laboratory animals during growth, gestation, lactation, maintenance, and reproduc- tion as recommended by the National Academy of Sciences (NAS 1974, 1977, 1978). It should be noted that a data gap exists in nutrient require- ments for animals involved in long-term studies beyond their reproduc- tive life span (NAS 1978). image: ------- Table 1. Aftroxtaurte Nitriot Coaqiositioi of EPA, NIH, AIN ud CoBSMfdal Laboratory Aiinal Diet FuiHuliBoai EPA" rodent diet Nutrients Crude protdn, rain., % Crude fat, rain., % Crude fibcr,% Ash, % Linoleic acid, % Total digestible nutrients (TON), % Amino acids, % Aiginine,% Cystine.% Glydne.% Histidine,% Isolcucinc, % Leucine,% Lyiine,% Metbionine, % Phenylalanine, % Threoninc,% Tryptophan, % Tyrosine,% Valine.% Minerals Calcium, % Chloride, % Chromium, ppro Cobalt, ppm Copper, ppm Fluoride, ppm Iodine, ppm Iron, ppm Magnesium, % Manganese, ppm Phosphorus, % Potassium, % Selenium, ppm Sodium, % Sulfur, % Zinc, ppm Vitamins Vitamin A, RJ/g Vitamin D, IU/g dVTocopherol (vitamin E), ppm Tniamine,ppm Riboflavin, ppm Niatin, ppm Pantotbcnic acid, ppm Choline, ppm Pyndoxine, ppm Folk acid, ppm Bntin,ppm Vitamin BIB ppm Vitamin K (Menadione), ppm Inoshol, ppm Vitamin C, ppm For autoclavabk diets: Vitamin A, IU/g Thiamin, ppm Vitamin K (Menadione), ppm min. 18.0 4.3 4.2 8.0 NG NG 0.95 0.27 1.00 0.38 0.95 1.50 0.90 0.38 0.90 0.65 0.20 0.60 0.95 1.15 NG NG 0.80 12.0 NG 1.85 345.0 0.20 140.0 0.90 0.80 0.1 0.33 NG 50.0 15.0 4.0 50.0 14.0 7.0 65.0 32.0 1900.00 10.0 2.0 0.3 0.03 3.0 NG NG 100.0 70.0 20.0 max. NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG 0.6 NG NG NG 75.00 10.00 NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG Agway* (Charles River) RMH3000 22.0 5.0 5.0 6.0 0.9 NG 1.63 0.44 1.35 0.51 1.10 1.83 1.41 0.42 1.13 0.91 0.27 0.83 1.15 0.97 0.15 NG 0.96 23.0 35.0 I.I 289.0 0.21 61.7 0.85 0.95 0.25 0.44 NG 84.0 20.229 1.045* 55.62 11.50 10.38 72.45 21.92 1542.00 8.84 1.67 0.40 0.06 0.97 NG NG 28.15 67.32 1.38 Purina' Rodent Lab Chow* 15002 20.0 4.5 5.5 (max.) 7.0 (max.) NG 77.0 1.13 0.27 0.86 0.49 1.03 1.58 1.18 0.43 0.88 0.78 0.24 0.60 1.05 0.90 0.47 2.09 0.6 13.3 NG 1.2 180.0 0.21 63.0 0.70 0.86 NG 0.30 NG 52.4 17.6 2.2 60.0 13.3 8.0 60.0 17.0 1800.0 6.0 4.0 0.13 0.02 NG NG NG 37.0 70.0 8.8 Purina' Guinea Pig Chow* |5026 18.0 4.0 16.0 (max.) 9.0 (max.) NG 67.6 1.08 0.28 0.87 0.43 0.96 1.46 0.95 0.40 0.89 0.71 0.27 0.59 0.95 1.1 0.30 6.01 0.38 21 NG 1.6 298.7 0.35 121 0.6 1.2 NG 0.32 NG 122 30.0 + 59.9^ 3.4 44.0 5.5 6.0 50.0 19.0 1850.0 4.0 4.2 0.30 0.01 NG NG 1000.0 NG NG NG NIH open'' formula mouse & rat diet 23.5 5.0 4.5 NG NG NG 1.25 0.35 1.10 0.50 1.10 1.80 1.20 0.50 1.10 0.90 0.25 0.75 1.20 1.20 NG NG 0.70 15 NG 1.80 250 0.15 100 0.95 0.80 NG 0.33 NG 45 15.0 4.0 35.0 14.0 7.0 80.0 20.0 2000.0 10.0 4.0 0.15 0.03 3.0 NG NG NG NG NG Purified diet' AIN-76* (rodents) 17+ 5 5 NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG NG 0.52 0.156 2.0 NG 6.0 NG 0.2 35.0 0.05 54.0 0.40 0.36 0.1 0.102 333.3 30.0 4.0 1.0 56.6 5.34 6.0 30.0 16.0* NG 7.0* 2.0 0.2 0.1 0.05 NG NG NG NG NG Purina' Canine Diet 15007 25.0 9.0 4.0 (max.) 10.0 (max.) NG 78.0 1.46 0.37 1.85 0.59 1.13 2.21 1.10 0.44 1.20 0.90 0.25 0.78 1.24 1.8 0.42 2.34 0.50 13.0 NG 1.70 241.4 0.24 85.0 1.0 0.71 NG 0.42 NG 48.1 40.0 4.4 44.0 9.8 4.5 60.0 20.0 2000.0 12.5 3.6 0.18 0.03 0.57 NG NG NG NG NG Purina' Rabbit Chow* 15322 16.0 2.5 18.0 (max.) 8.0 (max.) NG 66.0 0.90 0.25 0.77 0.40 0.82 1.30 0.78 0.35 0.80 0.64 0.23 0.75 0.84 0.95 0.40 4.56 0.38 13.0 NG 0.59 252.6 0.25 44.4 0.50 1.40 NG 0.30 NG 3X5 28 + 46^ 2.3 44.0 3.5 4.5 33.0 19.0 1600.00 4.5 1.2 0.12 0.01 NG NG NG NG NG NG NG — Not given. "Data taken from USEPA (1979). Data on ViL B^and Vit. A in the autoclavable diet have been converted into ppm and IU/g, respectively. Data taken from Agway Inc. (n.d). Rat, mouse, hamster 3000. "JData taken from Ralston Purina Company (1980). "Data taken from NIH (1980). *Data taken from AIN (1977) and NAS (1978). ' As carotene. 'Vitamin D,. "CasalL 'Hydrochloride. image: ------- Raf'd Growth, gestation Nutrient lactation Maintenance Protein, % Fat.% Digestible energy, kcal/g Fiber, % Linoleic acid, % Total digestable nutrients (TON), % L-Amino acids, % Arginine Asparagine Olutamic acid Histidine Isoleucine Leucine Lyiine Methionine Phenylalanine + tyrosine Praline Threonine Tryptophan Nonessential Minerals Calcium, % Chloride, % Cobalt, mg/kg Magnesium, % Phosphorus, % Potassium, % Sodium, % Sodium chloride, % Sulfur, % Chromium, mg/kg Copper, mg/kg Fluoride, mg/kg Iodine, mg/kg Iron, mg/kg Manganese, mg/kg Selenium, mg/kg Vanadium Zinc, mg/kg \2.W 5.00 3.8 NO* 0.6 NO 0.60 0.40 4.00 0.30 O.SO 0.75 0.70 0.601 0.80 0.40 0.50 0.15 0.59* 0.50 0.05 / 0.04 0.40 0.36 0.05 NO 0.03 0.30 5.00 1.00 0.15 35.00 50.00 0.10 NO 12.00 4.201 5.00 3.8 NO NO NO / / / 0.08 0.31 0.18 0.11 0.23 0.18 / 0.18 0.05 0.48" NO NO / NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Mouse" growth reproduction 12.5, 18.0 NO NO NO 0.3 NO 0.3 NO NO 0.2 0.4 0.7 0.4 0.5 0.4 NO 0.4 0.1 NO 0.4 NO NO 0.05 0.4 0.2 NO NO NO 2.0 4.5 n 0.25 25.00 45.0 o P f Golden Hamster"' 15.0 5.0 4.2 NO NO NO 0.76 NO NO 0.40 0.89 1.39 1.20 0.32 0.83 + 0.57 NO 0.70 0.34 NO 0.59 NO 1.1 0.06 0.30 0.61 0.15 NO NO NO 1.6 0.024 1.6 140.00 3.65 0.1 NO 9.2 Guinea pig"4 growth 18.0 <1« 3.0 10.0 NO NO NO NO NO NO NO NO NO NG NO NO NO NO NO 0.8-1.0 NG NO 0.1-0.3 0.4-0.7 0.5-1.4 NO NO NG 0.6 6.0 NO 1.0 50.0 40.0 0.1 NO 20.0 Rabbit* Dog*-" 20.0 4.5 NG NO 0.9 NO NG NO NO NO NO NO NO NO NG NO NO NO NO 1.0 NO NO 0.036 0.8 0.5 NG 1.0 NO NG 6.5 NG 1.39 54.0 4.5 0.10 NG 45.0 Growth 16.0 2.0 2.5 10-12 NG 65 NG NG NG 0.3 0.6 1.1 0.65 0.6 1.1 NO 0.6 0.2 NG 0.4 0.3 NO 0.03-0.04 0.22 0.6 0.2 NO NO NO 3.0 NG 0.2 o 8.5 NG NG o Maintenance 12.0 10 2.1 14 NG 5 NO NG NO NG NO NO NO NG NG NG NO NG NO 0 0.3 NO 0.03-0.04 0 0.6 0.2 NO NG NG 3.0 NO 0.2 0 2.5 NO NG o image: ------- Rat*-" Nutrient Vitamins A,IU/kg D, lU/kg E.IU/kg K,. Mg/kg Choline, mg/kg Folio acid, mg/kg Niacin, mg/kg Pantothenate, mg/kg (Calcium) Riboflavin, mg/kg Thiamin, mg/kg B,, mg/kg B,2, Mg/kg Biotin, mg/kg Inositol (myo-) Vitamin C, mg/kg Growth, gestation lactation Maintenance 4,000.00 1,000.00 30.00 50.00 1,000.0 1.00 20.0 8.00 3.00 4.00 6.00 50.0 k 1 m NG NG NG NG NG *NG M NG NG NG NG NG NG k 1 m Mouse"-" growth reproduction 500.0 150.0 20.0 3,000.00 600.0 0.5 1010 10.0 7.0 5.0 1.0 0.01 0.2 / m Golden Hamster"-' 3,634.00 2,484.00 3.00 4,000.00 2,000.0 2.00 90.0 40.0 15.0 20.0 6.0 10.0 0.6 100.0 m Guinea pig"4 growth 20,328.00 1,000.00 50.0 5,000.00 1,000.0 4.00 10.0 20.00 3.00 2.00 3.00 10.00 0.3 NG 200.00 Rabbit' Dog*-" 4,500.00 450.00 45.00 NG 1,100.0 0.16 10.3 9.0 2.0 0.90 0.90 20.00 0.09 NG m Growth 580.0 i 40.0 * 1,200.0 NG 180.0 NG NG NG 39.0 NG NG NG m Maintenance o t o k NG NG NG NG NG NG NG NG NG NG m "Data taken from NAS (1978). 'Data taken from NAS (1974). 'Data taken from NAS (1977) 'Based on 90% dry matter. 'Unless indicated, requirements listed for growth are the same for reproduction. When indicated, first value listed is for growth, second for reproduction. Values are estimated minimal requirements for conventional mice. /Estimates based on minimal amounts in diets adequate for growth for 6 weeks following weaning. 'Minimal requirements. *Nutrient levels selected to meet the requirements of the most demanding life cycle segments, i.e., rapid growth and lactation. 'Although not stated by author, presumably none required. 'NG - not given. *Not required, produced by bacterial synthesis in intestine. 'Not required under conventional laboratory conditions. "Dietary source not required. "Controversial whether or not required. "Required; no quantitative data given. 'No data; may be needed in ultraclean environments. «Unsaturated fatty acid. 'Probably required; amount unknown. 'As ideal protein. 'Partially supplied by L-cystine. "Mixture of glycine, L-alanine, and L-serine. image: ------- 2. NUTRITIONAL REQUIREMENTS FOR LABORATORY ANIMALS - COMPARISON OF THE DATA PROPOSED IN THE EPA GOOD LABORATORY PRACTICE STANDARDS WITH THOSE OF COMMERCIAL PRODUCERS. NIH. AND AIN Nutritional requirements vary from species to species and also with the physiological state of the animal within the same species. Guinea pigs and rabbits, for instance, can utilize cellulose to some extent (MAS 1977, 1978). This is reflected in the commercial diets for these animals. Purina guinea pig chow and rabbit chow include as much as 16 and 18% crude fiber, respectively (see Table 1). The NAS recommendation for fiber content of the guinea pig and rabbit diets is 10 and 10-12%, respectively (see Table 2). Of the laboratory animals under discussion, guinea pigs are unique in their absolute dependence on a dietary source of vitamin C. All commercial diets for guinea pigs are supplemented with vitamin C (see Table 1). Special and richer diets are often used in commercial reproducing colonies (NAS 1978) because, contrary to gen- eral assumption, diets that produce maximum postweaning growth are not necessarily adequate for maximum rates of reproduction. It is worthwhile to point out that the Appendix B of the TTSEPA (1979) was probably written with respect to the rat, mouse, and hamster, although these animal species are not specified. It apparently was not written for rodents in general since vitamin C, which is essential in the diet for the guinea pig, is not included in the list of dietary requirements. Also, Purina rodent chow #5002 (Ralston Purina Company 1980) is a misnomer, since this chow, lacking in vitamin C, is not intended for the guinea pig. The proper designation for these chows should mention the names of the animals for which they are intended, as exemplified in NIH (1980) and Agway Inc. (n.d.) (see Table 1). As Table 1 indicates, commercial feed manufacturers are generally in good agreement with the dietary requirements proposed by EPA for rodent diets (a 10% variance is permitted by EPA). Notable exceptions are manganese (140 ppm vs 61.7 ppm for Agway rat-mouse-hamster 3000 diet) and pantothenic acid (32 ppm vs 17 ppm for Purina rodent chow), vitamin A [100 lU/g (30 ppm) vs 28.15 TO/g (8.44 ppm) for Agway's rat- mouse-hamster autoclavable diet], and vitamin K (20 ppm vs 1.38 ppm for Agway's rat-mouse-hamster autoclavable diet). Table 2 shows the estimated nutrient requirements for the species of concern in this study as recommended by the National Academy of Sci- ences (NAS 1974, 1977, 1978). These data support the use of special diets in production colonies. Comparison of the nutrient requirements listed in Table 2 with those proposed by EPA and the dietary composition of commercially available feeds (see Table 1) indicates that for most nutrients, the EPA and commercial levels are generally higher than the estimated requirements recommended by the NAS. This is because the nutrient concentrations in commercial diets and those proposed by EPA are formulated to compensate for losses during the manufacture and storage as well as for differences in biological availability of nutrients. image: ------- 3. EVALUATION OF DIFFRBEMT TYPES AND FORMS OF DIETS The two criteria most widely used for judging the adequacy of a diet are: (1) growth curves and (2) reproduction data (i.e., litter size, average weight at birth, average number weaned, average weaning weight, and estrus cycle). Normal growth data for hamsters, rats, and mice are presented in Table 3, and growth curves for rabbits, guinea pigs, and dogs are shown in Figures 1, 2, and 3, respectively. Newberne (1982) has suggested that the study of the subchronic effect of a com- pound for 90 days should be used to establish dose levels that will result in no more than 10% depression in body weight gain. Reproduction and life span data for these animal species are shown in Table 4. Vari- ous types of laboratory animal diets are described in the following sec- tions. A carefully designed long-term tozicity experiment with labora- tory animals should take into account the possibility of induction of enzymes by the dietary components and/or contaminants. Such enzymes may have the potential to alter the tozicity of the chemical under investi- gation (Newberne 1975). This topic is discussed in Section 9.4. 3.1 Types of Diets - Advantages and Disadvantages 3.1.1 Natural-Ingredient Diets These diets are economical and are the most widely used. They are made from appropriately processed whole grains such as wheat, corn, oats, or commodities that have been subjected to limited amounts of refinement such as fish meal, soybean meal, or wheat bran. Some of the disadvantages are: the necessity for commercial manufacture; the inabil- ity to completely control the nutrient concentrations; batch-to-batch variation in nutrient concentration; difficulty in altering composition to study the effect of the absence of a particular nutrient; and the potential for contamination with pesticide residues, heavy metals, or other agents that might alter the response to experimental treatment (NAS 1978). 3.1.2 Purified Diets Diets formulated with refined ingredients such as casein, sugar, starch, vegetable oil, and cellulose are known as purified diets. Some of the advantages are that (1) ease of preparation in the laboratory, (2) capability for duplication of nutrient concentrations or alteration for induction of nutritional deficiencies or excesses, and (3) low potential for chemical contamination. Disadvantages include being more expensive than natural-ingredient diets and not palatable to all species of animals (NAS 1978). Purified diets can be both open formula and closed formula diets. 3.1.3 Chemically Defined Diets These diets are prepared by mixing chemically pure compounds such as amino acids, sugars, triglycerides, essential fatty acids, inorganic salts, and vitamins. Two advantages are ease of preparation in the image: ------- Table 3. Nonul Growtfc Rate of the HuMtcr, Rat, ud Mow Range and average body weights (g) as a function of age (days) Animal species Outbred Golden Hamsters CnRHG (SYR) Male Female Rats: Sprague-Dawley Male Female Rats: Fischer Male Female Inbred mouse: Male Female 21 range 29-51 30-50 50-61 45-63 24-39 22-39 7.7-15.8? 7.5-14.8* av. 40 40 46" 44" NG NG 11.0 10.2 28 range 32-71 31-69 71-114 71-130 28-56 35-51 11.3-20.5* 9.9-18.6° av. 49 44 75" 64° 53° 44° 15.7 14.2 42 range NGC NG NG NG NG NG 14.4-28.7? 13.1-25.8* av. NG NG NG NG NG NG 21.0 18.6 58 range 86-98 85-104 206-259 188-205 155-209 105-156 16.1-30.8* 15.1-28.96 84 av. 92 95 236° 185° 160° 123° 24.1 21.5 range 99-109 103-127 317-385 231-283 183-238 112-175 NG NG av. 104 115 302a 210a 213° 145" NG NG 112 range NG NG NG NG NG NG 23.2-35.7? 21.1-37.6° 168 av. NG NG NG NG NG NG 30.2 28.1 range 128-142 150-167 436-482 275-301 312-380 203-238 NG NG av. 141 158 365" 230" 256° 162° NG NG "Average body weights are averages of figures given by two different suppliers of the two strains. Vr Range of averages for 26 inbred mouse strains. cNot given. Source: NAS (1978). image: ------- ORNL-DWG 82-17970 oo h- LU 10 9 8 7 6 5 4 3 2 1 0 I I I I I T AGE: days 30 40 50 60 70 weeks 6 8 10 months 1 2 80 90 100 110 120 130 140 150 160 170 180 190 200 12 16 21 25 3456 Figure 1. Growth curve for rabbit (New Zealand White). (Source: Dutchland Laboratories, Inc. 1982.) image: ------- 900 ORNL-DWG 82-17971 800 I— 700 I— 600 h- * 500 I— o 400 h- 300 I— 200 I— 100 I— 6 8 10 AGE (weeks) 12 14 16 Figure 2. Growth data for guinea pig (Ft. Detrick Dunkin Hartley) (Source: Dutchland Laboratories, Inc. 1982.) image: ------- ORNL-DWG 82-17972 61.7 59.9 COCKERS BEAGLES SCOTTIES FOX TERRIERS DACHSHUND BOSTONS PEKINGESE 0 2 10 12 14 16 18 20 22 24 26 28 30 MONTHS Figure 3. Growth curves for fifteen breeds of dogs. 1974, p. 1.) (Source: NAS 10 image: ------- Table 4. Reproduction" and Life Spai* Data on Selected Laboratory Animate Weight Adult male Adult female At birth Breeding Age Male Female Breeding Data Estrus cycle Gestation Weaning age Litter size Breeding life - Male Female Rebreeding Mating Life span Hamsters 85-1 10 g 95-1 20 g 2g 60 days/85-1 10 g 60 days/95-120 g 4-15 days 15-19 days 20-24 days/35 g 6-10 1 year 10-12 months/4-5 litters 4-6 days Pair 1 yr. av., 2 yr. max. Mice 20-40 g 25-40 g 1-5 g 50 days/20-35 g 50-60 days/20-30g 4-5 days 17-21 days 16-21 days/10-12 g 1-23/av. 12 18 months 6-10 litters immediately Pair or colony; 1 male, 3 females 1.5 yr. av., 3 yr. max. Rats 300-400 g 250-300 g 5-6 g 100 days/300 g 100 days or 200 g 5 days 20-22 days 21 days/40-50 g 8-12 1 year 1 year immediately Pair or colony 1 male, 3-4 females 3 yr. av., 4 yr. max. Dogs 13.-18.5 kg (beagle) 13.5-16 kg 350-450 g 10-12 months 9-12 months bi-annually 60-65 days 6-8 weeks 4-8 6-14 years 6-10 years next heat period 1 male, up to 40-60 females 15 yr. av., 35 yr. max* Guinea pigs 1000-2000 g 850-900 g 100 g 90-150 days/550 g 90-150 days/500 g post partum 16-18 days 59-67 days 10 days/250 g 1.6/av. 4 5 years 4-5 years immediately Colony: 1 male, 3-10 females 3 yr. av., 7 yr. max. Rabbits 4.5-5 kg 4.5-6.5 kg 100 g 6-7 months/4 kg 5-6 months/4.5 kg polyestrus 30-32 days 8 weeks/1.8 kg 1-18/av. 8 1-3 years 1-3 yean 35 days Pair. Mate in bucks cage; 1 male, 6-10 females 6 yr. av. 15 yr. max. "Data taken from Ralston Purina Company (n.d.). *Data taken from Agway Inc. (n.d.). image: ------- laboratory and strict control of nutrient concentrations. The fact that these diets are too expensive for general use and that availability of nutrients may be altered by oxidation or interactions among nutrients represents the principal disadvantages (NAS 1978). 3.1.4 Open Formula Diets These are diets of known ingredient composition and are manufac- tured from natural, purified, or chemically defined ingredients. Specifications for open formula diets not only state the amount of each ingredient to be used but also define the minimum acceptable nutrient composition of ingredients to be used. Advantages include the fact that the diet can be procured by competitive bidding, resulting in lover cost compared with the brand name diets under sole source negotiated con- tracts, and that the investigator has the option to make alterations in nutrient composition to satisfy specific program objectives (NIH 1980). 3.1.5 Closed Formula Diets Most widely used closed formula diets are natural-ingredient diets, but the ingredient compositions are well-kept secrets of the manufactur- ers. In general, most laboratory animals thrive on these diets. Some disadvantages are that these diets introduce an unknown element (such as nature and composition of ingredients) that can be critical to the interpretation of the results in a particular investigation and that the diets can be procured only through negotiated sole source contracts. A list of ingredients as well as the nutrient composition of the diet is provided by the manufacturer (NIH 1980). These diets have extensive historical background data. Recently, Newberae (1982) reported wide variation in the nutrient composition of closed formula diets, and toxic component composition of natural ingredient diets from commercial sources has been reported (Table 5). 3.1.6 Quasi-Open Formula Diets The closed formula diet can be procured through competitive bidding by specifying a list of acceptable ingredients and thereby constitutes a quasi-open formula diet. However, the manufacturer is not obligated to use all of the listed ingredients (NIH 1980). 3.2 Forms of Diets Diets in various physical forms have been used for laboratory animals. These include (a) meal, (b) pelleted, (c) crumbled, (d) extruded, (e) baked, (f) flaked, (g) semimoist or gel forms, and (h) liquid. For a detailed discussion of these different forms of diet, the reader is referred to the document published by the National Academy of Sciences (NAS 1978). The pelleted, extruded, gel, and liquid forms of diets are discussed below. The pelleted diet is the one most widely used for feeding labora- tory animals. During the pelletization process, meal is heated to 180- 190°F for 30 seconds to form dense compressed pellets that are easier to 12 image: ------- Table 5. Concentrations of selected nutrient and toxic components in random samples of rodent natural product diets Component Requirements, rat" Diet analysis* Minerals Calcium (%) Phosphorus (%) Magnesium (%) Copper (ppm) Zinc (pppm) Amino acids Tryptophan (%) Methionine (%) Phenylalanine + tyrosine (%) Vitamins Vitamin A (mg/kg) Vitamin D (lU/kg) Riboflavin (mg/kg) Toxicants Aflatoxins (ppm) Nitrates (ppm) Nitrosamines (ppb) JV-dimethylnitrosamine JV-nitrosopyrrolidine Lead (ppm) 0.56 0.44 0.04 5.60 13.3 0.17 0.67 0.89 0.67 1108 0.5-4.0 0.5, 0.27, 1.67 1.90, 0.68, 0.13 0.54, 0.02, 0.23 3.8, 6.9, 65 20, 17, 10, 60 0.10, 0.39, 0.08 0.13, 1.21, 0.22 2.01, 1.05, 1.82 2.10, 0.31, 3.75 987, 1360, 5100 12, 0.3, 0 0.01, 0.20, 0.12, 0.0 90, 5, 10, 18, 32 8, 0, 32, 18 0, 7, 5, 16 0.8, 1.8, 4.2, 2.5 "Requirements according to the National Research Council, 1978a. * Each line represents different samples from the same manufacturer. Analysis conducted at MIT over a period of 15 years. Source: Newberne 1982. 13 image: ------- handle and store. Wastage is minimized, and the daily dietary intake of the test animals is relatively easy to measure. However, if admin- istered in the diet, the substance under study has to be incorporated in the meal before the pellets are made. During the manufacture of the extruded diet, the meal is subjected to moist heat (350-400°F for 1 minute). This treatment partially cooks the starch and makes the diet highly palatable to some animals, e.g., nonhuman primates, dogs, cats. However, other laboratory animal species waste a lot of feed, and frequent feeding is necessary because of the low diet density. Diets in semimoist or gel forms are produced by adding water, agar, gelatin, or other jelling agents, to meal. These diets are more palat- able than dry rations and more convenient for incorporating the sub- stances to be tested. They also allow efficient measurement of food consumption. However, because these diets are susceptible to microbial growth, they must be kept frozen or refrigerated and feeding must be at frequent intervals. Large quantities are thus difficult to handle. Following the pioneering liquid diet research of Lieber and DeCarli (1966), Bio Serve Inc. has been offering isocaloric liquid diet formula- tions for rats, mice, and dogs. This diet offers an excellent delivery option in toxicity studies of volatile test substances. 3.3 Autoclaving of Diets Extent of loss of heat sensitive components of the diet, e.g., vitamins, is partly determined by the procedure used during autoclaving. It is perhaps desirable to recommend a standardized procedure in the EPA Good Laboratory Practice (USEPA 1979) for autoclaving laboratory animal feed. One procedure, used by Agway (Agway Inc. n.d.), is outlined below. PROCEDURES FOR STERILIZATION OR PASTEURIZATION OF R-M-H 3500 Since autoclaves differ, these procedures will be outlined in three (3) generalized phases so that the operator of any par- ticular autoclave may successfully sterilize or pasteurize the formula based on operational procedures peculiar to that autoclave. PHASE I. CREATE VACUUM AND DEVELOP HEAT Adjust jacket pressure to normal. Place volume of pelleted feed in autoclave according to its capacity, then pull a vacuum up to 25 to 29 inches for approximately 15 minutes. Afterward, slowly meter in steam while maintaining a vacuum of 10 to 15 inches. The interval of time required should be sufficient to bring the pellets to a heat of 180 to 190°F (10 to 15 minutes). Meter steam in slowly so that the pellets are brought to the above temperature and yet moisture is not 14 image: ------- permitted to condense on or within the pellets. Experimenta- tion may be required. PHASE II. PASTEURIZATION Tarn off vacuum pump. Introduce full volume of steam. This should elevate the temperature within the pellets to 215 to 220°F. This will require approximately 20 to 25 minutes from the time steam pressure reaches 15 Ibs. PHASE II. STERILIZATION Turn off vacuum pump. Introduce full volume of steam. This should elevate the temperature within the pellets to 250 to 260°F. This will require approximately 20 to 25 minutes from the time steam pressure reaches 15 Ibs. PHASE III. EXHAUST AND DRY Open chamber exhaust and vent to 2 to 5 Ib. of steam pres- sure. Turn on vacuum pump pulling 20 to 25 inches to remove steam from the autoclave. The drying cycle (approximately 30 minutes depending on capacity of vacuum pump and quantity of feed) may be adjusted according to desired dryness. Stop vacuum pump and introduce bacteriologically filtered air into the chamber. 3.4 Discussion Highly purified diets and chemically defined synthetic diets are the diets of choice when true and reliable standard diets are required over an indefinite period. However, these diets have two main disadvan- tages — they are expensive and they are not consumed well by all species. The use of the natural-ingredient diets of known composition (open formula) has been advocated (AIN 1977). Admittedly there is sea- sonal and year-to-year variation in the composition of the natural ingredients but by specifying the standards of the quality of the ingredients, the variability can be kept to a minimum. The list of approved ingredients should be revised from time to time to allow inclu- sion of new, improved, or more economical substitnents. However, as Knapka (1974) has pointed out, "the use of open formula rations could result in a liability without adequate control of product quality and sanitation of manufacturing facilities. Quality assurance at NIH is obtained by requiring that the facilities used to manufacture the ration be in compliance with the sanitation standards in the National Insti- tutes of Health Standard Number 1" (NIH 1980). It is imperative that other animal feeds containing drugs (e.g., cattle feed, poultry feed) should not be manufactured on the same premises where laboratory animal feed is manufactured to avoid possible cross contamination. The closed formula diet on the other hand has a history of accept- able performance although the investigator is left in the dark so far as the composition of the natural ingredients is concerned. These diets 15 image: ------- are also generally more expensive than the open formula diets. The manufacturers closely guard the secrecy of the diet formula, and they are free to alter the composition of the ingredients according to their availability and prices (AIN 1977). It will be very difficult to unearth any interaction between dietary components and the test sub- stance in experiments using closed formula diets. Although the open formula diet need further development and improvement, compared with the closed formula diet, the ability to control the diet composition will certainly aid in obtaining sound and reproducible animal experiments. The National Institutes of Health are currently using open formula diets for the rat, mouse, hamster, guinea pig, and rabbit. The food for dogs, however, is purchased via an advertised contract using a specification for a quasi-open formula diet. Manufacturers may use any combination of the listed ingredients to produce a diet with the indicated nutrient concentrations (NIH 1980). Recent data indicate that highly purified diets are not infallible, although the use of such diets for definitive studies has been strongly recommended (Newberne 1982). The AIN-76A™ diet, for example, has been reported to cause severe periportal lipidosis in the livers of male F- 344 or CD rats (Hamm et al. 1982). The AIN-76711 diet causes nephrocal- cinosis in female Sprague-Dawley rats (Nguyen 1982). The presence of traces of solvents used in the purification of some of the ingredients in semisynthetic diets may also cause complications. Assuming that a perfect diet, free from contaminants, preserva- tives, etc., is used in long-term toxicity testing with animals, one cannot help but raise the question of how relevant will data from such tests be for assessing the risk to man, who is normally fed his share of preservatives, pesticide residues, etc. It appears that the practical solution to this problem is to establish adequate quality control over nutrients and contaminants and rigid protocols in natural ingredient diets to ensure reproducibility of results free from any interference due to dietary factors, no matter whether closed formula or open formula diets are used in the experiment. Newberne and McConnell (1981) have also strongly recommended surveillance over both contaminants and nutrients in natural product as well as semipurified diets. This has become extremely important in view of the fact that closed formula diets have been found to differ considerably in the composition of nutrients and contaminants (Newberne 1982, Table 5). 16 image: ------- 4. DIETARY INTAKE During the long-term toxicity test, it is not unusual for an animal to lose or gain weight at a lower than normal rate. The weight loss or reduced rate of weight gain may be due to low dietary intake or to the toxic effect of the chemical or to both. Excessive weight in laboratory animals should also be avoided since the response to chemical stress of an overweight animal is likely to be different from that of a normal animal. It is critical, therefore, to monitor the food intake of the test animals. Equal food intake between control and test animals can be achieved by pair feeding (Newberne et al. 1978). Most laboratory animals are coprophagous, which introduces an unknown element in the diet through ingestion of soiled bedding. This factor however, can be minimized by the use of screen-bottomed rather than solid-bottomed cages (Newberne 1978). The effects of dietary restrictions on immunity and aging have been reported by Weindrnch and Makinodan (1981). Undernutrition without mal- nutrition has led to increased life-span and lower spontaneous cancer incidence in mice (Weindruch and Walford 1982). What is true for mice may be true for humans, but it may not be wise to do long-term toxicity studies with laboratory animals on restricted diets since this will introduce an additional factor in extrapolating the results of animal experiments to human exposure. Besides, administration of restricted diets to a large number of laboratory animals is expensive and diffi- cult. 17 image: ------- 5. VITAMINS AND MINERALS USED IN ANIMAL DIET FORMULATIONS 5.1 Vitamins The guinea pig is unique among the laboratory animal species under discussion (mouse, rat* hamster, guinea pig, rabbit, and dog) in its dietary requirement for vitamin C (MAS 1978). Most other animals can synthesize sufficient amounts of vitamin C to meet their daily require- ment so the vitamin does not need to be added to their diet. Unfor- tunately, the ascorbic acid in the pelleted feed is not stable. This phenomenon has been investigated at Hoffmann-La Roche (n.d.) using cry- stalline ascorbic acid and ethylcellnlose-coated ascorbic acid. Fifty- one percent of the ethylcellulose-coated ascorbic acid is retained after storage for 3 months at room temperature whereas only 27% of the uncoated crystalline ascorbic acid was retained under the same condi- tions. Ethylcellulose-coated ascorbic acid is used in NIB open formula diets (NIH 1980). Additional ascorbic acid supplementation of the feed is necessary if the feed is to be used during the second 3-month period of storage. The preservative ethozyquin is used as a stabilizer for vitamin A in both the NIH open formula diet and commercial diets (see Sect. 7.4). Preservatives and their use for this purpose should be considered for inclusion in the proposed EPA guidelines. Present EPA guidelines (USEPA 1979) do not permit the use of any preservative in the animal feed. Inositol is not normally required in the diet of laboratory animals; however, the National Academy of Sciences recommends a level of 100 ppm in the diet of the golden hamster (MAS 1978). Inositol may also be required under special circumstances, e.g., rodents kept on antibiot- ics (NAS 1978). Table 6 lists sources of vitamins included in the EPA GLP (USEPA 1979), NIH open formula rations for mouse/rat and germ-free mouse/rat (NIH 1980), AIN-76™ purified mouse and rat diet (AIN 1977), Purina Rodent Chow #5002 and autoclavable Rodent Chow #5014 (Ralston Purina Company 1980), and Agway R-M-H 3000 and autoclavable R-M-H 3500 (Agway Inc. n.d.). Choline bitartrate and thiamine hydrochloride have been used in the AIN-76TM purified diet in place of choline chloride and thi- amine mononitrate, respectively. The latter two sources are included in the EPA GLP (USEPA 1979) and are also constituents of commercial diets. Consideration should be given to the inclusion of choline bitartrate and thiamine hydrochloride in the proposed guidelines (USEPA 1979). Purina Rodent Chows #5002 and #5014 apparently do not include any source of biotin and probably depend on the natural occurrence of biotin in feed ingredients. 5.2 Minerals Table 6 lists sources of minerals included in the EPA GLP (USEPA 1979), NIH open formula rations for mouse/rat and germ-free mouse/rat (NIH 1980), AIN-76TM purified mouse and rat diet (AIN 1977), Purina 18 image: ------- Tabte 6. Vitamins, Minerals, umi Aafanal Diet FormbtW NIH* A. Yitamims Vitamin A acetate Vitamin A palmitate EPA GLP* ; Mouse/rat diet •j j mouse/rat diet ^ AIN-76* purified diet'' - Rodent Autoclavable chow |5002 chow |5014 R-M-H 3000 j "<*T R-M-H 3500 (autoclavable) ' Vitamin D activated animal sterol (03 j j •/•/•/ J j j Menadione activity « j s s dl,a-Tccopherol acetate -j j j j Choline bitartrate J Choline chloride j j s j j j j Folic acid j j j j j •/ •/ •/ Niacin ^ j j s j s -j •> j ^ image: ------- Rodent Chow #5002 and autoclavable Rodent Chow #5014 (Ralston Purina Company 1980), and Agway R-M-H 3000 and autoclavable R-M-H 3500 diets (Agway Inc. n.d.). As Table 6 indicates, the sources of these minerals other than those specified in the USEPA (1979) include the following: copper oxide, cnpric carbonate, ferrous carbonate, ferric citrate, zinc sulfate, zinc carbonate, monocalcium phosphate, manganous carbonate, potassium iodate, citrate, sulfate, sodium selenite, and chromium potas- sium sulfate. In addition to the latter five sources mentioned above, monocalcium phosphate, zinc carbonate, ferric citrate, and cupric car- bonate are used only in the AIN-76^M purified mouse amd rat diet. A purified diet requires supplementation with trace elements because essentially none are provided in the major ingredients. Although chromium is required in the purified AIN-76^ diet and in the NAS recom- mended guinea pig diet (Tables 1 and 2 respectively), it is not used in Ralston Purina or Agway Inc. commercial diet formulations (Table 1). Silica is used as an anticaking agent in autoclavable diets - Purina Rodent Chow #5014 and Agway R-M-H 3500. Most of the diet formulations except the purified diet depend on the natural presence of selenium in the feed ingredients and do not include any extraneous selenium in the form of sodium selenite, although the addition of extraneous selenium has been permitted by the EPA as an exception to the Delaney clause (see Sect. 10.3). Copper oxide, cupric carbonate, ferrous carbonate, iron ammonium citrate, zinc sulfate, zinc carbonate, manganese carbonate, and potassium iodate are included in the 6RAS list (USFDA 1980a) (Table 7). Consideration should be given to expansion of the list of mineral sources in USEPA (1979) to include these different mineral forms and silica, allowing some flexibility in the diet formulation. 20 image: ------- Table 7. Trace minerals included in the GRAS List0 Element Source compounds Cobalt Copper Iodine Iron Manganese Zinc Cobalt acetate Cobalt carbonate Cobalt chloride Cobalt oxide Cobalt sulfate Copper carbonate Copper chloride Copper gluconate Copper hydroxide Copper orthophosphate Copper oxide Copper pyrophosphate Copper sulfate Calcium iodate Calcium iodobehenate Cuprous iodide 3,5-Diiodosalicylic acid Ethylenediamine dihydroiodide Potassium iodate Potassium iodide Sodium iodate Sodium iodide Thymol iodide Iron ammonium citrate Iron carbonate Iron chloride Iron gluconate Iron oxide Iron phosphate Iron pyrophosphate Iron sulate Reduced iron Manganese acetate Manganese carbonate Manganese citrate (soluble) Manganese chloride Manganese gluconate Manganese orthophosphate Manganese phosphate (dibasic) Manganese sulfate Manganous oxide Zinc acetate Zinc carbonate Zinc chloride Zinc oxide Zinc sulfate "These substances added to animal feeds as nutritional dietary supplements are generally recognized as safe when added at lev- els consistent with good feeding practice. Source: USFDA (1980a). 21 image: ------- 6. INGREDIENTS USED IN LABORATORY ANIMAL DIETS All of the ingredients listed in Appendix B of the EPA Good Labora- tory Practice Standards, July 26, 1979 (TJSEPA 1979), are used in the NIH and commercial diet formulations (see Table 8). However, some ingredients are not included in the above-mentioned list but are used in both NIH and commercial diet formulations. These include animal fat protected with butylated hydroxyanisole (BHA), cane molasses, meat and bone meal, and dried skimmed milk. In addition, the commercial diets include casein, dried whey, ground beet pulp, and wheat germ meal, which are not used in the NIH diet (Table 8). The NIH dog diet formulations allow the use of the following preservatives in various ingredients: BHA, propylene glycol, propyl gallate, and citric acid (NIH 1980). Animal feeds containing animal fat without preservative have a shelf life of about 12 weeks when stored at room temperature (personal commun- ication from Dr. John C. Chah, Ziegler Bros. 1982). However, when soy oil is used as a source of fats, it contains enough natural antioxidants (tocopherols) to obviate the necessity of adding any extraneous preser- vative. From the available information it seems that none of the feed manufacturers have any control over the supply of the food ingredients, although this is where the most effective control on the major source of contaminants in the feed can be exercised. It would seem advisable to include in a given set of guidelines the specifications (grade, etc.) for individual ingredients. To illustrate the point consider the NAS specification for wheat (NAS 1974). As many as six different types of wheat have been listed as common dog food ingredients: (1) wheat, durum, triticum durum. (2) wheat, hard red spring, triticum aestivum, (3) wheat, hard red winter, triticum aestivum, (4) wheat red spring, triti- cum aestivum, (5) wheat, soft, triticum aestivum, and (6) wheat, soft red winter, triticum aestivum. These vary considerably in their nutri- tional quality (e.g., protein content varies from 12 to 16.3% on dry basis). The NTH open formula diet avoids all these uncertainties by specifying hard winter wheat (NIH 1980). The other source of contaminant control is the guidelines for proper sanitation in facilities used to manufacture the ration (NIH 1980, Appendix A, Standard 1) . 22 image: ------- Table 8. Ingredients Commonly Used in Laboratory Animal Diets u> . .. ingrcaicnt Agway R.M-H 3000 Purina 5002 certified rodent chow Purina canine diet #5007 Purina guinea pig chow #5026 NIH open formula mouse & rat diet Alfalfa meal Brewers dried yeast Corn gluten meal Dicalcium phosphate Fish meal Ground whole wheat Ground #2 yellow shelled corn Ground whole oats Ground limestone Salt Soy oil Soybean meal Wheat middlings Ground beet pulp Wheat germ meal Cane molasses Dried whey Dried skimmed milk Casein Animal fat & BHA Meat & bone meal A. Ingredients listed in the EPA GLP (USEPA 1979) •/ / v v image: ------- 7. CONTAMINANTS AND PRESERVATIVES IN ANIMAL FEEDS 7.1 Introduction Contaminants in laboratory animal feeds arise from biological as well as nonbiological sources. Biological contaminants originate from microorganisms growing on plants in the field, grain, and prepared feed. These are mainly estrogens, aflatozins, and trichothecene toxins. Non- biological contaminants are usually pesticide residues and nitrosamines. When laboratory animals consume any contaminant residue daily in their diet, they are in effect chronically exposed to a considerable amount of the contaminant over their lifetime; for instance, when a rat is exposed to 100 ppb of lindane in its daily dietary intake of 12 g, the total chronic exposure over its life-span of 3 years amounts to about 1.3 mg. These dietary contaminants have the potential for affect- ing the outcome of long-term animal experiments in various ways. They may act as carcinogens, cocarcinogens, cancer promoters, teratogens, mutagens, or general toxicants. They may also induce the production of microsomal enzymes that may affect the metabolic pattern and inhibit neoplasia (see Sect. 9.4). In either case, both inhibitors and enhanc- ers of toxicity are present in the natural ingredient diet. These diets thus differ from semipurified diets in the way they 'permit' an animal to respond to a test substance, usually in the animal's favor (Newberne and McConnell 1981). This protective action may have a good or bad effect on experimental results, depending on the purpose of the study. This effect extends to spontaneous as well as chemically induced tumors. If the objective is to accumulate animal data for extrapolation to assess the toxicity to man, the presence of these dietary contaminants in the animal feed may not be bad since it is practically impossible for humans to consume food completely free from pesticide residues. Preservatives are chemicals deliberately added to animal feed to increase its shelf life and preserve the easily oxidizable vitamins present in the feed. The following subsections will describe in more detail biological contaminants, nitrosamines, and preservatives and will examine the differences between EPA's proposed contaminant tolerances for rodent diets and the contaminant levels found in commercial rodent feeds. 7.2 Biological Contaminants 7.2.1 Estrogens Table 9 lists three estrogens that may occur in animal feed. These are formed when a crop is infected with fungi in the field or during storage. The three estrogens in Table 9 show a low level of estrogenic activity in the following order: genistein > coumesterol > zearalenone. Therefore, they are usually not of much concern in animal feed manufac- ture. However, when animals are used for studies on reproduction, the estrogenic activity of the feed should be assayed. 24 image: ------- Table 9. Estrogens in Animal Feed Estrogen Chemical structure Estrogenic activity compared with DBS (on oral dosing) Occurrance Stability Genistein <10-> Soybeans (6 Mg DES/kg) Heat stable Coumestrol ro <35 X 10-' Alfalfa, soybeans; alfalfa infested with fungal Heat stable pathogens (115 ppm) Zearalenone OH o CH3 620 X 10~s Possibly produced on corn during storage particu- larly when infested with Fusarium graminearum Heat stable Diethylstilbestrol Source: Compiled from Stob (1973). image: ------- 7.2.2 Aflatoiins Aflatozins are a family of 10 or more brightly fluorescing furano- coumarin compounds. The most prominent of these are Bl, B2. Gl, and G2- 62 and 62 are partially reduced (dihydro) forms of Bl and Gl, respec- tively. 6 0 10 OCH3 Aflatoxin Bi(B2) OCH3 Aflatoxin G!(G2) Aflatozins are produced by Aspergillus flavus, a common mold grow- ing on a variety of food materials including corn, soybean, peanuts, rice, and wheat. Although aflatozin Bl is fairly stable to heat, a reduction of aflatozin Bl in peanut meal from 7000 to 340 ug/kg has been achieved by autoclaving the moist meal (60% moisture content) at 15 Ib pressure (120°C) for 4 h. The photodecomposition of aflatozin is known to occur at fairly low light levels (Wilson and Hayes 1973). Aflatozin Bl appears to be the most potent hepatocarcinogen known (Miller 1973). Moderate to high incidences of hepatomas and hepatocel- lular carcinomas have occurred in rats continuously administered afla- tozin in the diet at concentrations as low as IS ppb. Experimental evi- dence suggests that aflatozin Bl needs to be metabolically activated to be carcinogenic in vivo (Miller 1973). The ubiquitous presence of the mold Aspergillus flavus indicates the improbability of complete elimination of aflatozin contamination from animal feed; however, high humidity and a temperature of 25°C have been found to be conducive to the growth of this mold. Like most fungi Aspergillus flavus is highly aerobic. The FDA has lowered the maximum amount of aflatozins permissible in food products as methods of detec- tion and control have improved. The upper limit has been set at 20 ppb (total, Bl, B2« Gl, and G2) in feeds [EPA proposed guidelines (USERA 1979) specify 5 ppb] and 0.5 ppb aflatozin MI in milk (USFDA 1980b). Aflatozin MI is a metabolite of aflatozin Bl (4-hydrozy derivative of aflatozin Bl) and has been identified in the milk of cows ingesting aflatozin Bl. 26 image: ------- 7.2.3 Tricothecene Toxins These my co tor ins comprise a family of closely related sesquiter- penoids produced by various species of fungi such as Fusarium, Myrothe- cium. Trichoderma, Trichothecium. Cephalosporium, Verticimonosporium and Stachybotvs and are collectively called trichothecenes after tricothe— cin, the first isolate, discovered in 1949 by Freeman and Morrison. Fusarium tricinctum and Fusarium roseum are the two most important fungi as far as animal feeds are concerned. The former causes moldy corn (or hemorrhagic) disease and the latter causes vomiting and feed-refusal in animals. Both can grow on corn, barley, and rye. T-2 toxin, HT-2 toxin, and diacetoxyscirpenol are produced by Fusarium tricinctum. and deoxynivalenol, nivalenol, zearalenone and an unknown factor are pro- duced by Fusarium roseum. The chemical structure, LD50» natural occurrence, and biological effects of these toxins are described in Table 10. It has been suggested by Schoental (1981) that some of the incon- sistencies in epidemilogical and experimental studies on the role of various types of fats in the etiology of cardiovascular disorders and certain tumors may be related to the presence of adventitious contam- inants (e.g., tricothecenes) in the fats. Various toxicological (e.g., vomiting in ducklings), cytological (e.g., cytotoxicity to HeLa cells) and biochemical (e.g., inhibition of protein synthesis in rabbit reticnlocytes) assays have been developed for tricothecenes. A sensitive radioimmunoassay capable of detecting 1 and 2.5 ppb of T-2 toxin in wheat and corn, respectively, has been recently reported (Lee and Chu 1981). 7.3 Nitrosamines Nitrates and nitrites have been used for the preservation of fish and meat, often in conjunction with sugar, spices, polyphosphates, a-tocopherol, and sodium ascorbate. The use of ascorbic acid is rather fortuitous since it greatly reduces the chance for nitrosation of amines in the stomach by nitrous acid. Besides protecting meat from spoilage by bacteria such as Clostridium botulinum. nitrite imparts red color to the meat. It reacts with myoglobin to form nitrosylmyoglobin, which during cooking becomes denatured; the color is retained because of the formation of nitrosylmyochrome. N-Nitroso compounds are produced in food principally from the reac- tion of naturally occurring secondary amines with nitrites that may have been added to the food or formed from bacterial reduction of nitrate. The most common volatile nitrosamine present in food is N-nitroso- dimethylamine (NDMA). N-Nitrosopyrrolidine and N—nitrosomorpholine have also been detected. The latter, however, has been shown to be formed principally from morpholine used as a corrosion inhibitor in boilers when food is processed by steam in a smokehouse. 27 image: ------- Table 10. Fnsarinm Toxins" rO CO Toxin Formula LDso' Foods Naturally Contaminated with Mycotoxin Comments Fusarlum trtclnctum Diacetoxyscirpenol T-2 toxin HT-2 toxin* Fusarium roseum Nivalcnol Deoxynivalenol" (vomitoxin) Zearalenone Structure I RLR2.R3-H R4,Rj,—OAc RS-OH Structure I Rl,R3-H R2—3-methylbutyryloxy R4,R6-OAc RS-OH Structure I R,,R3=H R2—3-methylbutyryloxy R4-OAc R$.R6-OH Structure I RI.=O R3,R4.R5,R image: ------- Volatile nitrosamine contamination of laboratory animal diets has been investigated by Edwards et al. (1979). Low levels of N-nitro- sodimethylamine (1-4 ppb) have been found in most of the commercial pel- leted diets. Higher levels (5-50 ppb) of N-nitrosodimethylamine (NEWA) have been detected in three out of seven samples of NIH open formula rat and mouse ration, and low levels of N-nitrosopyrrolidine (0.3 ppb to 2.1 ppb) have also been detected in these samples. Silverman and Adams (1983) investigated the presence of N-nitrosamines in laboratory animal feed and bedding used by NIH using a gas chromatograph with a thermal analyzer. N-Nitrosodimethylamine was detected in all samples of NIH-07 meal (1.2-6.9 ppb) and 62% of NIH-07 pellets (0.6-35.2 ppb). It was also detected in 66% of closed formula meal (1.9-45.1 ppb) and 83% of closed formula pellets (0.2-21.3 ppb). Low concentrations (1-4 ppb) were found in samples of hardwood chip and corncob bedding. It appears that the poor quality of the fish meal is responsible for the higher nitrosamine content of NIH-07 diets. The elevated temperature created during the regrinding of food pellets to produce meal diets may be a contributing factor to the formation of nitrosamin.es. In addition, atmospheric nitrogen oxides may be absorbed and subsequently react with secondary or tertiary amines on the greater surface area of the meal diet. It has been demonstrated that as little as 10 ppb of MDMA included in the water supply of Strain A/J mice (tumor-prone) during the whole fetal, weaning, and post-weaning period (up to 22 weeks of age) causes a tripling in the lung tumor incidence from 8% to 23% (Edwards and Fox 1979). However, it is likely that even 50 ppb of NDHA may not cause a significantly increased incidence of cancer in non-tumor-prone mice. Synergistic effects of MDMA with 3-methylcholanthrene have been demon- strated at a high NDHA level but not at the low levels found in labora- tory animal diets. However, it seems advisable to keep the level of MDMA in laboratory animal diets as low as possible to avoid any poten- tial problem. At one time it was thought that an appreciable quantity of volatile nitrosamines present in the feed is lost during the pelletization pro- cess (Enapka 1979), but this view is now discounted (personal communica- tion from Enapka 1982, NIH, and Shelton 1982, Ralston Purina). One com- menter has reported (comments respective to proposed EPA guidelines) that antoclaving of the feed reduces the nitrosamine content. Although there appears to be no documentation of this observation in the open literature, investigating ways and means for reducing the nitrosamine content of fish meal used in the production of laboratory animal feed would certainly provide valuable information. The detection and estimation of nitrosamines in animal feed at the ppb level is carried out using a thermal energy analyzer nitrosamine detector, interfaced with a gas or liquid chromatograph (Edwards et al. 1979). 29 image: ------- 7.4 Preservatives Two of the most widely used preservatives in laboratory animal feed are ethoxyquin and butylated hydrozyanisole (BHA). The former is used as an antiozidant in vitamin A formulations (She1ton 1982, NIB 1980). Ethoxyquin retards the oxidation of carotene, xanthophylls, and vitamins A and E and prevents organic peroxide formation in canned pet food. It is also added to dehydrated forage prepared from alfalfa, barley, clover, bermnda grass, corn, fescue, oats, orchard grass, sorghums, wheat, etc., for this same reason. The tolerance limit for ethoxyquin in animal feed has been set at 150 ppm by the FDA (USFDA 1980a). Vitamin A stabilized with ethoxyquin is manufactured by Hoffmann-La Roche, New Jersey. In a micronized form, it contains gelatine and other additives, as well as ethoxyquin, 7%, and vitamin A acetate, 650,000 lU/g. This calculates to 8 ppm of ethoxyquin in feed containing 75 III of vitamin A/g (EPA proposed maximum in rodent feed, see Table 1), well below the tolerance limit set by the FDA. However, the purified diet described for rabbits in the Nuritional Requirements for Rabbits (MAS 1977) permits a rather high concentration of ethoxyquin (250 ppm). Soybean oil contains sufficient amounts of natural antioxidants, making further addition of preservatives unnecessary; however, whenever animal fat is used in feed formulation, BHA has been frequently used as a preservative. For instance, Purina Rodent Chow #5002 does not contain animal fat and consequently no BHA. The Agway RMH 3000 diet, Purina Canine Diet #5007, Purina Guinea Pig Chow #5026, and the NIH dog diet formulation contain animal fat and unspecified amounts of BHA. There is no evidence of storage of BHA in fatty tissues in rats ingesting BHA and no evidence of chronic toxicity in rats fed up to 91% (1000 ppm) in the diet for 2 years (Patty's Industrial Hygiene and Toxicology 1981a). Both ethoxyquin and BHA have been found, however, to be potent inducers of hepatic epoxide hydratase when administered in the food of rats at 0.1% for 14 days (Kahl and Klaus 1979), potentially resulting in enhanced metabolism of a toxicant. Appreciable improvement (20-30%) in the stability of vitamin C in pelletized feed during storage has been claimed for the ethylcellulose- coated vitamin C preparation of Hoffmann-La Roche. The NIH open formula diets specify the use of this stabilized form of vitamin C. However, the product has yet to gain popularity with feed manufacturers. Ethyl- cellulose presumably affords protection by acting as a moisture barrier (Hoffmann-La Roche n.d.). It is very inert physiologically and has been found to be nontoxic (Patty's Industrial Hygiene and Toxicology 1981b). The NIH open formula diet also permits the use of propylene glycol, propyl gallate, and citric acid as preservatives in animal feed (NIH 1980); however, the allowable concentrations of these ingredients have not been specified. 30 image: ------- 7.5 Tolerance Limits of Contaminants and Preservatives in Animal Feed Table 11 lists the tolerances proposed by EPA for certain contam- inants and preservatives in rodent diets (USEPA 1979). For comparison the maximum concentrations of feed contaminants considered acceptable for natural ingredient rations for rodent diets of the National Center for Tozicological Research (NCTR) (NEC 1976), and the maximum levels of contaminant permitted (guaranteed analysis) in the Agway R-M-H (rat- mouse-hamster) 3000 diet and guinea pig diet (Agway Inc. n.d.) and in the Ralston Purina Guinea Pig Chow #5026 and Rodent Chow #5002 (Ralston Purina Co. 1980) are also provided. Also included in Table 11 are the maximum permitted contaminant levels in several nonrodent diets. When the data in this table are compared, the following becomes evident: 1. The EPA maximum tolerances exceed those of NCTR for all con- taminants with the exception of estrogenic activity (1 ppb vs 2 ppb) and PCB (50 ppb vs 500 ppb). 2. The Agway R-M-H 3000 and guinea pig diets are identical with respect to maximum allowable levels of contaminants whereas the maximum permissible concentrations of malathion, DDT, cad- mium, mercury, and PCB are lower in these diets than in the Purina Rodent Chow #5026. 3. The guaranteed maximum levels of contaminants permitted in commercial rodent diets exceed the proposed EPA maximum toler- ance for lindane, heptachlor, DDT (Purina chows only), dield- rin, cadmium, mercury (Purina chows only), and PCB (Purina chows only). 4. No preservatives are allowed according to the proposed EPA guidelines, whereas all the commercial producers use bntylated hydroxyanisole in diets containing animal fats. Besides the contaminants listed in Table 11, the feed industry also mon- itors the level of heptachlor epoxide (a metabolite of heptachlor), endrin, chlordane, benzene hexachloride, toxaphene, phorate, diazinon, disulfoton, methyl (ethyl) parathion, endosulfan, ethion, and carbo— phenothion. The maximum levels of these contaminants permitted in Agway and Purina rodent feed are indicated in Table 12 and, as shown, are quite similar between the two producers. (Table 11 was restricted to a comparison of those contaminants listed in the proposed EPA guidelines - a list which the EPA recognizes as being inconclusive.) In addition to the contaminants listed in Tables 11 and 12, a host of chemicals (primarily pesticides) are identified by the FDA (USFDA 1980a) and EPA (USEPA 1981) as potential contaminants in animal feeds. Table 13 lists some of the chemicals more likely to be found in labora- tory animal feeds, along with their uses and tolerance levels. Neither the EPA nor the FDA tolerance listed is that for the finished feed but rather for raw commodities and feed ingredients, respectively. Pyreth- rins in conjunction with piperonyl bntoxide have been reported to be 31 image: ------- Table 11. Contaminants and Preservatives in Laboratory Animal Feeds EPA proposed tolerance for rodent diet Contaminants min. Aflatoxins (total) ppb Estrogenic activity (DBS eq), ppb Lindane, ppb Heptachlor, ppb Malathion, ppm DDT (total), ppb Dieldrin, ppb Cadmium, ppb Arsenic, ppm Lead, ppm Mercury, ppb Selenium, ppm 0.1 PCB, ppb Nitrosamines, ppb Antibiotics Preservative max. 5* 1 20 20 2i5 100* 20 160 1.0 1.5 100 0.6 50 10 0 0 National Center' for Toxicological Research rodent diet 1.00 2.00 10.00 10.00 0.50 150.00 10.0 50.0 0.25 1.00 50.0 0.5 500.00 NG NG NG AgwayCl^ (Charles River) R-M-H 3000 5.0 NG 50 30 0.3 100 30' "<• 200 1.0 1.5 100 0.2 50 NG NG BHA+* Agwayc Purina Purina (Charles River) Agway Rodent Chow* Guinea Pig Guinea (Charles River) #5002 Chow* #5026 Pig Diet Rabbit Diet 10.0 NG 50 30 0.5 150 50.0 500 1.0 1.5 200 NG 150 5-12> NG NG 10.0 NG 50 30 0.5 150 50.0 500 1.0 1.5 200 NG 150 NG NG BHA+* 5.0 NG 50 30 0.3 100 30' 200 1.0 1.5 100 0.2 50 NG NG BHA+* 5.0 NG 50 30 0.3 100 30' 200 1.0 1.5 100 0.2 50 NG NG NG Agwayc Purina*' Purina (Canine Diet Rabbit Chow* Canine Diet respond 2000 #5322 #5007 5.0 NG 50 30 0.3 100 30' 200 1.0 3.0 100 0.2 50 NG , NG BHA+* 10.0 NG 50 30 0.5 150 50.0 500 1.0 1.5 200 NG 150 NG NG NG 10.0 NG 50- 30 0.5 150 50.0 500 1.0 3.0 200 NG 150 NG NG BHA+* NG — Not given. "All data are maximum allowable levels unless otherwise stated. *Data taken from USEPA (1979). cData taken Agway Inc. (n.d.). Data taken from Ralston Purina Company (1980). 'Data taken from NRC (1976). •'Rat-Mouse-Hamster diet. * Animal exposure to aflatoxins in food is limited to the level of 20 ppb (total BI, 82, GI, 62) under the current FDA regulations (USFDA, 1981). *The permissible limit in animal feed is 5 ppm under the USFDA (1980). +Aldrin. •'Personal communication from Purina. BHA, butylated hydroxyanisole. image: ------- Table 12. Pesticides and Contaminants in Laboratory Animal Feeds Common name Aldrin + dieldrin Heptachlorepoxide Endrin Chlordane BHC Toxaphene Phorate, Thimet Diazinon Disulfoton Methyl(ethyl)parathion Endosulfan, Thiodan Ethion Carbophenothion, Trithion Chemical name 1 ,2,3,4, 1 0, 1 0-hexachloro- 4-endo-S,8-dimethanonaphthalene (aldrin) Hexachloroepoxyoctahydrc-endo, endo-dimethanonaphthalene 1,2,4,5,6,7,8,8-Octachlor- 2,3,3a,4,7,7a-hexahydro- 4,7-methanoindane Benzene hexachloride Chlorinated camphene O.O-Diethyl S-{(ethyIthio)- methyl)phosphorodithioate O.O-Diethyl O-(2-isopropyl-4- methyl-6-pyrimidinyl) phosphorothioate O.O-Diethyl S-{2-(ethylthio)- ethyl}phosphorodithioate O,O-Dimethyl)ethyl) o-p-nitrophenyl phosphorothioate 6,7,8,9,10,10-Hexachloro 1 ,5,5a,6,9,9a-hexahydro- 6,9-methano- 1 ,4,3-benzo(e)- dioxathiepin-3-oxide O.O.O.O-Tetraethyl S.S-methylene bisphosphorodithioate S-{(p-chlorophenylthio)methyl| O,O-diethyl phosphorodithioate Use Insecticide (soil insects) Metabolite of heptachlor Insecticide Insecticide (termites) No longer produced or sold for domestic use in the United States Insectcide (soybeans, sorghum, and peanuts) Insecticide (alfalfa, corn, wheat, etc.) Insecticide and pesticide Insecticide and acaricide Insecticide Insecticide Insecticide Acaricide Tolerance, p] Agway (Charles River) R-M-H 3000* 0.03 0.03 0.03 0.05 0.05 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.3 ?ma Purina rodent chow* #5002C 0.05+0.05 0.05 0.05 0.05 NG NG 0.5 0.5 0.5 0.5+0.5 0.5 0.5 0.5 NG = Not given. "Values are maximum allowable levels. Data taken from Agway (n.d.). Rat-Mouse-Hamster 3000. cData taken from Ralston Purina Company (1980). 33 image: ------- Table 13. Pesticides IB Laboratory Animal Feeds CO -is Tolerance (ppm) Common name Acephate Aldicarb Aluminum phosphide NF Banvel Benomyl Bcntazon, Basagran Butachlor Chlordimeform Chlorpyrifos 2,4-D Dalapon Daminozide, Alar Demeton, systox Dialifor, Torak NF NF NF NF Chemical name O,S-Dimethyl acetylphosphoramidolhioare 2-Methyl-2(methylthio)proionaldehyde O-(methyl-carbamoyl )oxime Aluminum phosphide 4-Amino-6-(l,l-dimethylethyl)- 3-(methylthio)- 1 ,2.4-triazin-S (4H)-one 2-Methoxy-3,6-dichlorobenzoic acid or 3,6-dichloro-o-anisic acid Methyl 1 -(butylcarbamoyl)-2-benzimidazole carbamate 3-( 1 -Methylethyl)- 1 H-2. 1 ,3-benzothiadiazin- 4(3H)-one 2,2-dioxide 2-Chloro-2'.61-diethyl-N- ., v (butoxymethyl)acetanilide N'-(4-Chloro-c-tolyl)-N,N- dimethylformamidine O,O-Diethy!O-(3,5,6-trichloro-2-pyridyl) phosphorothioate 2,4-Dichloro-phenoxy acetic acid 2,2-Dichloropropionic acid Butanedioic acid mono (2,2-dimethyl hydrazide) Mixture of O.O-diethyl O-|2-(ethylthio)ethyl| phosphorothiate and O.O-diethyl S-|2-(ethylthio)ethyl) phosphorothioate S-(2-Chloro- 1 -phthalimidoethyl) O.O-diethyl phosphorodithioate N>-(2,4-Dimethylphenyl)-N-[{(2,4-dimethylphenyl) unino)methyl]-N-methylmethanimidamie 2-Ethoxy-2,3-dihydro-3,3-dimethyl-S-benzofuranyl methanesulfonate S-[2-(EthylsuIfinyl)ethyl] O,O-dimethyl phosphorothioate O-Elhyl O-[4-(metbylthio)-phenyl] S-propyl phosphorodithioate Use Insecticide Systemic insecticides, acaricide, nematicide for soil use Fumigant insecticide Herbicide Herbicide Fungicide Herbicide Herbicide Insecticide Insecticide Herbicide Growth regulation Plant regulator Systemic insecticide Insecticide Insecticide Herbicide Insecticide Insecticide Feed Soybean Cottonseed hulls Soybean Bulk grain Barley, wheat Sugar cane molasses Tomato pomace Corn, wheat, sorghum Dehydrated citrus pulp Citrus fruit Soybean Rice, bran Cottonseed hulls Cottonseed Sugar beet molasses Sugar beet pulp, dried Sorghum milling fractions Sorghum grain Barley, oats, rye, wheat Sugarcane molasses Dehydrated citrus pulp Citrus frut, grain crops (excl. wheat) Dried tomato pomace Peanut Sugar beet pulp Sugar beet Dehydrated citrus pulp Citrus fruit Dehydrated citrus pulp Citrus fruit Dehydrated citrus pulp Sugar beet molasses Sugar beet Sorghum Cottonseed hulls Cottonseed USFDA" 4C 0.3 0.1' 3.0 0.3 2.0 h 50.0 NA 0.5 10.0 3.0 11.0 1.5 2.0 5 20.0 600.Q 90.0* 5.0 5.0 15.0 3.0 0.5 2.0 1.0 USEPA* 1.0 0.02 0.01 0.75 NG NO h 10.0 0.05 NG 5.0 NG NG 0.75 0.5 0.5 30.0 0.5 0.75 3.0 0.3 NG 0.5 image: ------- T«Me 13. (continued) Tolerance (ppm) Common name Chemical name NF Hexakis (2-methyl-2-phenylpropyl) distannoxane Magnesium phosphide Magnesium phosphide Methanearsonic acid Methanearsonic acid Paraquat l,l'-Dimethyl-4,4'-bipyridinium dichloride Phosalone, Zolone O,O-Diethyl S-{-(6-chloro-2-oxo-benzoxazolin- 3yl)-methyl)phosphorodithioate Picloram, Tordon 4-Amino-3,5,6-trichloropicolinic acid Piperonyl butoxide a-{2-(2-l-Butoxyethoxy)ethoxy)-4,5- methylenedioxy-2-propyItoluene Plictran Tricyclohexyl hydroxystannane Profenofos, Curacron O-(4-Bromo-2-chlorophenyl)-O-ethyl S-propyl phosphorothioate Ui Propanil 3',4'-Dichloropropionanilide « Pyrethrins Simazine 2-Chloro-4,6-bis(ethylamino)-s-triazine TDE (DDD) 1 , 1 -Dichloro-2,2-bis(p-chlorophenyl)ethane NF S.S.S-Tributyl phosphorotrithioate Tutane Sec-butylamine Zn ion + maneb NA - not applicable; NF - not found; NO - not given. "tolerances in feed ingredients, USFDA (1980). Tolerances in raw commodities, USEPA (1981). ^Soybean meal. Peanut meal. 'AS2C-3. * Cation. *No tolerance given. Use Insecticide Fumigant insecticide Herbicide Herbicide Insecticide Herbicide Synergist wih pyrethrin (insec- ticide); treatment of bags used for packaging food Acaricide Insecticide Herbicide Insecticide; treatment of bags used for packaging food Herbicide Insecticide Herbicide Fungicide Fungicide Feed Dehydrated citrus pulp Citrust fruit Not specified Barley, corn, cottenseed Cottonseed hulls Cottonseed Sugarcane mollasses Sunflower seed hulls Sunflower seed Dehydrated citrus pulp Citrus fruit Barley, oats, wheat Not specified Barley, rye, wheat Dried citrus pulp Citrus fruit Soybean Cottonseed hulls Rice milling fraction Rice Not specified Barley, rye, wheat Sugarcane molasses Alfalfa Dried tomato pomace Tomato Cottonseed hulls Cottonseed Dehydrated cirus pulp Citrus fruit Barley, oats, rye, wheat Wheat Corn grain USFDA" 7.0 O.le 0.9/ 3.(X 6.0* 12.0 3.0 10.0 8.0 — h 6.0 10.0 1.0 1.0 100.0 6.0 90.0 20.0 USEPA* 4.0 0.1 0.7 2.0 3.0 0.5-1.0 20.0 2.0 NO NO 2.0 3.0 15.0 7.0 0.25 30.0 1.0 0.1 Use indicated in Farm Chemicals Handbook (1981). image: ------- used for the treatment of bags used for packaging, primarily to discourage insect infestation of the stored food (USFDA 1980a). The current manufacturing practice probably does not make use of any insec- ticide treatment of the bags used for packaging animal feed, as exempli- fied by the Ralston Purina Company (Jon Ford 1982). 7.5.1 Considerations for Establishing Tolerances for Contaminants Three of the most important considerations in setting tolerance limits for contaminants in animal feed are analytical capability, bio- logical no-effect levels, and interactions between dietary components and contaminants. There is no point in setting a tolerance limit below the detection limits of the currently available analytical techniques. Normal screening levels of detection (not necessarily the detection limit) of different contaminants have been reported by Greenman et al. (1980) (see Table 14). Assay of contaminants at such low levels pro- vides a challenge to the analytical chemist. Therefore, reproducibility of various assays and variation from laboratory to laboratory need to be investigated. The open literature contains biological effect and no- effect data for some contaminants (see Tables 15 and 16). Such data need to be developed for other contaminants of interest. Interaction between dietary components and toxicants is an active area of research. A few representative examples are given in Table 17. Many dietary con- stituents such as BHA are known to affect the metabolism of chemical carcinogens by microsomal mixed-function oxidases in liver, lung, intes- tine, and other tissues (Schrauzer 1977). Any synergism, antagonism or initiator-promotor activity of the contaminant is certain to influence the outcome of long-term toxicity studies. 7.5.2 Suggested Changes in the Tolerance Limits for the Contaminants Listed in the EPA GU > 7.5.2.1 Aflatoxins In harmony with the current FDA guidelines (USFDA 1980b), con- sideration should be given for setting a tolerance of 0-20 ppb. 7.5.2.2 Estrogenic activity This contaminant has not posed any serious problem in the past so far as animal feed manufacture is concerned (Greenman et al. 1980). The estrogenic activity has been found to be so low that it is no longer monitored at NIH (personal communication from J. Enapka 1980). The NIH bioassay procedure uses 4 ppb of DBS (diethylstilbestrol) as a positive control, and animal feeds are regarded to have positive estrogenic activity only when they exhibit higher activity than the standard con- taining 4 ppb of DBS. The tolerance level possibly should be raised to 4 ppb. 7.5.2.3 Lindane Even though a concentration of 0-50 ppb of lindane is allowed in animal feeds by many producers, most of the commercial feed analyzed by 36 image: ------- Table 14. Normal Screening Levels of Detection Analyte Normal screening level of detection Reference Aflatoxin 5 ppb Horwitz (1970c) Stubblefield et al. (1967) Beljaars et al. (1975) Przybylski (1975) Pesticides and PCBs DDT Dieldrin Heptachlor Lindane PCBs Malathion As Cd Ca Pb Se Hg Cu Zn Vitamin A Vitamin Bt Fat Protein 5 ppb 3 ppb 3 ppb 3 ppb 20 ppb 50 ppb 50 ppb 5 ppb 1000 ppb 500 ppb 40 ppb 20 ppb 20 ppb 20 ppb 10 lU/g 5 ppm 0.5% 0.5% Bowman and Beroza (1970) Bowman et al. (1971) McMahon and Sawyer (1975a, 1975b) Horwitz (1970d) Burchfield and Johnson (1965) Brody and Chaney (1966) Hundley and Underwood (1970) Horwitz (1970b) J. Assoc. Off. Anal. Chem. (1973a) Heckman (1968) Horwitz (1970a) Horwitz (1970b) Horwitz (1970b) J. Assoc. Off. Anal. Chem. (1973b) Hoffman et al. (1968) J. Assoc. Off. Anal. Chem. (1971) Horwitz (1970a) Horwitz (1970a) Heckman (1968) Bowman and King (1973) Bowman (1973) Horwitz (1970a) APHA (1965) USEPA (1969) Source: Adapted from Greenman et al. (1980), p. 237. 37 image: ------- Table 15. Biologically Effective Concentrations of Selected Organic Feed Contaminants Dietary concentration (ppm) Compound Species Aflatoxin Mouse Rat DDT Mouse Rat Dieldrin Mouse Rat Heptachlor Mouse Rat Lindane Mouse Rat Malathion Rat End point Liver lipids Liver tumors Liver hyperplasia Liver tumors Reproduction Neoplasia Liver tumors Liver tumors Lung tumors Liver morphology Microsomal enzymes Microsomal enzymes Reproduction Mortality Liver tumors Microsomal enzymes Microsomal enzymes Liver weight Brain lesions Liver weight Liver tumors Microsomal enzymes Microsomal enzymes Tumor induction and liver morphology Microsomal enzymes Microsomal enzymes and liver weight Liver weight Cholinesterase Cholinesterase and EEG DMBA-induced tumors Effect 0.114 1 0.001 0.015 3 2 250 10 5 2.5 1 2.5 10 0.1 5 5 1 0.34 1 5 5 2 50 20 100 1000 380« 250 No effect 0.057 - - 3 20 - 1 2 0.2 - 1 - 1 2 0.1 0.02 - 1 1 50 20 2 100 - - Reference Lindenfelser et al. (1976) Newberne and Butler (1969) Wogan et al. (1974) Wogan and Newberne (1967) Tarjan and Kemeny (1969) Tarjan and Kemeny (1969) Tomatis et al. (1972) Terracini et al. (1973) Shabad et al. (1973) Laug et al. (1950) Gillett (1968) Kinoshita et al. (1966) Virgo and Bellward (1975) Walker et al. (1972) Walker et al. (1972) Gillett and Chan (1968) Den Tonkelaar and Van Esch (1974) Walker et al. (1969) Harr et al. (1970) Epstein (1976) Epstein (1976) Gillett and Chan (1968) Den Tonkelaar and Van Esch (1974) Weisse and Herbst (1977) Den Tonkelaar and Van Esch (1974) Pelissier and Albrecht (1976) Cecil et aL (1974) Hazelton and Holland (1953) Desi et al. (1976) Silinskas and Okey (1975) "Estimated equivalent. Source: Greenman et al. (1980), p. 242. 38 image: ------- Table 16. Biologically Effective Concentrations of Selected Heavy Metals Element Species End point As Mouse Reproduction Growth and mortality Mortality Mammary tumors" Rat Growth Cd Mouse Reproduction Mortality Rat Renal vasculature Hypertension Pb Mouse Mortality Growth Antibody formation Rat 5-Aminolevulinate dehydratase Mortality Hg Mouse Mortality and growth Rat Hypertension Behavior and learning Kidney ultrastructure Se Mouse Reproduction Growth and mortality Mammary tumors' Rat Growth, mortality, and tumors AAF-induced tumors'* Hepatitis Dietary or water concentration (ppm) Effect No effect 5 5 5 10 62.5* 31.25* 10 5 0.2 1 5 5 14 10 1 5 5 5 2.5 2" 2* 3 3 2 2 0.5* 0.1* 2.5* 0.5* Reference Schroeder and Mitchener (197 la) Schroeder and Mitchener (197 la) Schroeder and Balassa (1967) Schrauzer and Ishmael (1974) Bryon et al. (1967) Schroeder and Mitchener (197 la) Schroeder et al. (1964) Fowler et aL (1975) Perry and Erlanger (1974) Schroeder et al. (1964) Schroeder and Mitchener (197 la) Roller and Kovacic (1974) Hubermont et aL (1976) Schroeder et al. (1965) Schroeder and Mitchener (197 la) Perry and Erlanger (1974) Olson and Boush (1975) Fowler (1972) Schroeder and Mitchener (197 la) Schroeder and Mitchener (1972) Schrauzer and Ishmael (1974) Schroeder and Mitchener (1971b) Harr et al. (1973) Harretal. (1973) "Decreased incidence, increased tumor growth rate. • Mixed in the diet; all others were administered 'Decreased tumor incidence. Increased latent period. Source: Greenman et al. (1980), p. 241. in water. 39 image: ------- Table 17. Interaction of Dietary Factors with Toxicants Dietary factors Toxic agents Biological response Reference Protein Deficiency Fat Increased intake Addition of herring oil and linoteic acid High fat, low in choline, methionine, folic acid High fat Lipotropes Low lipotropes Low lipotropes Low lipotropes, high fat Carbon tetrachloride Dimethylnitrosamine Pesticides Mercuric chloride Dimethylnitrosamine Dimethylnitrosamine Chloroform Carcinogens 7,12-Dimethylbenzanthracene Azaserine Aflatoxin 7,12-Dimethylbenzanthracene Low lipotropes, high fat N-2-fluorenylacetamide Vitamins Vitamin A Vitamin A Ascorbic acid a-Tocopherol (Vit. E) Polynuclear aromatic hydrocarbon Benzo(a)pyrene Secondary amines 3-Methylcholanthrene Reduced toxicity (rats) Increased toxicity Reduced toxicity (rats) Reduced carcinogenicity Reduction in spontaneous hepatomas (rats) Increased sensitivity Maximum synthesis of cytochrome P-450 Enhanced liver tumor induction More mammary tumors (rats) than in animals on regular diets Increased susceptibility to liver carcinogenesis Increased hepatotoxicity More mammary tumors (rats) than in animals on regular diets More mammary tumors (rats) than in animals on regular diets Inhibits induction of squamous metaplasia and squamous cell tumors Enhanced induction of respiratory tract tumors Reduces nitrosation Reduces sarcomas in mice McLean and Verschurn (1969) Boyd (1969) Swithin and Yagi (1958) Swan and McLean (1968) Silerstone and Tannenbaum (1951) Goldschmidt et al (1939) Marshall and McLean (1969) Rogers et al. (1974), Rogers (1975) Corrou and Khor (1970) Shinozuka et al (1978) Newberne et al (1968) Newberne and Rogers (1976) Newberne and Rogers (1976) Saffiotti et al (1967) Cone and Nettesheim, 1973 Smith et al (1975) Smith, Rogers, and Newberne (1975) Fan (1973) Haber (1962) image: ------- Table 17. (continued) Dietary factors Toxic agents Biological response Reference Minerals Arsenic High Zinc Selenium Selenium Low iron Low magnesium, potassium Butylated hydroxyanisole, butylated hydroxy toluene High fiber diet Fiber Butylated hydroxyanisole, ethoxyquin Molybdenum (Mo) Cadmium (Cd), Mercury (Hg) Carbon tetrachloride (CC^) Benzo(a)pyrene (BP) Aflatoxin, estrogens Carcinogen/procarcinogens Benzo(a)pyrene (BP) 7,12-Dimethylbenzanthracene Dimethylnitrosamine Decreases cancer susceptibility; an essential micronutrient? Increased toxicity to Mo (rats) Decreased toxicity to Cd, Hg Reduces the toxicity of CCLj which catalyzes lipid peroxidation. Increased hepatic mixed function oxidase activity Decreased oxidase activity Decreases the carcinogenicity of BP Reduces the toxicity and carcinogenicity of the agents Decreases transit time, alters the bacterial flora in the gut, reduces incidence of colon cancer Decreases the carcinogenicity of BP Frost (1978) Brinkman and Miller (1961) Farkas(1978) Fodor and Kemeny (1965) Becking (1976) Becking (1976) Carr (1982) Carr (1982) Burkitt(1971, 1976) Wallenberg (1979) "No toxicant interaction. image: ------- Greenman et al. (1980) during 1974-78 showed much less than 20 ppb (see Table 18). Feed manufacturers should be able to comply with the currently set level of 20 ppb. No change, therefore, seems warranted. 7.5.2.4 Malathion Consideration should be given to reducing the tolerance level of malathion from 2.5 ppm to perhaps 0.5 ppm since it appears the feed industry can easily comply (see Tables 11, 18). 7.5.2.5 Cadmium Commercial samples (see Table 18) contained much less than the pro- posed tolerance of 160 ppb of Cd during 1974-78 (Greenman et al. 1980). However, it should be noted that the Agway and Ralston Purina certified diets may contain 200 ppb and 500 ppb, respectively (see Table 10). If there is any problem meeting this tolerance level (160 ppb), Cd-rich ingredients in the feed should be identified and replaced. No change seems warranted. 7.5.2.6 Arsenic Until the status of As as a micronutrient is established, the current tolerance limit of 1.0 ppm seems appropriate (see Sect. 10.2 for further discussion). 7.5.2.7 Selenium Consideration should be given to reducing the upper level of Se to 0.4 ppm, since Se at a 0.5 ppm level has been reported to increase the latent period for 2—acetylaminoflnorene-induced tumors (Greenman et al. 1980) (see Sect. 10.3 for;further discussion). 7.5.2.8 Preservatives Addition of ethozyquin, ethylcellnlose, and butylated hydrozyan- isole (BHA), should perhaps be permitted in the feed in keeping with the current practice for the preservation of vitamin A, vitamin C and animal fat, respectively. This will also permit an increase in the shelf life of the feed to six months instead of three months as currently stipu- lated in the EPA GLP (USEPA 1979). Hoffman-La Roche formulations of protected vitamin A contain 7% of ethozyquin; at the highest level of vitamin A permissible in the feed (75 HJ/g) in the proposed EPA guidelines (USEPA 1979), the concentration of ethozyquin is 8 ppm. This is well below the tolerance limit set by the FDA (150 ppm). A tolerance limit of 10 ppm for ethozyquin in animal feed would seem satisfactory. The amount of crude fat used in animal diet formulations can be ezpected to be as high as 9% (see Table 1). USFDA (1980a) allows a BHA 42 image: ------- Table 18. Results of Analyses of Laboratory Animal Diets CO Annual average* Analyte- Lindane (ppb) Heptachlor (ppb) Malathion (ppm) DDT (ppb) PCB (ppb) Dieldrin (ppb) Cd (ppb) As (ppm) Pb (ppm) Hg (ppm) Se(ppm) Ca(%) Cu (ppm) Zn (ppm) Vitamin A (lU/g) Vitamin B, (mg/100 g) Protein (%) Fat (%) 1974 2.20 2.64 0.11 14.0 0.5 4.9 79.4 0.038 0.39 0.036 0.30 - - - 36.95 7.36 - - 1975 5.96 4.10 0.38 151.61 0.5 12.15 84.93 0.01 0.89 0.037 0.43 - - - 27.68 8.91 - - 1976 («=54) 1.81 1.11 0.62 13.93 0.5 1.21 86.30 0.38 0.42 0.023 0.42 1.25 12.99 108.07 49.08 9.03 24.43 5.33 1977 0.84 0.32 0.10 18.10 19.85 1.43 93.15 0.32 0.34 0.019 0.22 1.16 15.20 106.66 40.31 9.46 23.80 5.72 1978 0.26 0.02 0.10 10.11 16.80 0.18 87.27 0.01 0.55 0.02 0.30 1.08 16.72 110.10 38.73 9.61 24.50 5.5 Mean («=148«) 1.67 1.07 0.33 27.7 8.7 2.35 87.3 0.25 0.47 0.024 0.34 1.16 15.0 108.2 41.6 9.1 24.2 5.54 Accumulated 5-yr value SD 3.6 2.24 0.53 48.4 15.0 4.6 33.2 0.28 0.38 0.018 0.15 0.18 2.8 9.7 36.9 1.25 2.4 0.58 Maximum value 40 12.3 2.4 309.1 55.0 26.3 168.0 0.92 1.92 0.16 0.66 1.67 22.0 140 439 13.1 44.4 6.6 Minimum value 0.02 0.02 0.005 0.15 0.5 0.08 5.0 0.01 0.02 0.007 0.04 0.82 6.9 78 11.5 5.7 21.7 4.0 CV< 215 209 161 175 172 197 38 112 81 75 44 16 19 9 89 14 10 10 •In addition to the analyses reported here, each lot was analyzed for aflatoxin and estrogenic activity, the results of these analyses were always less than the detectable concentration (5 ppb). 'Five new analytes were added to the surveillance list in 1976; this accounts for the voids (indicated by dashes) in the table. 'Results are reported for 148 production lots of autoclavable laboraory diet. 'Coefficient of variation. Source: Greenman et al (1980), p. 240. image: ------- level in the diet of 0.02% of the fat content. Therefore, consideration for a tolerance limit for BHA of 20 ppm in the finished diet should be given. Both BHA and ethoxyquin have been shown to be good inducers of enzymes and inhibit the carcinogenic effect of B(a)P and DMBA; however, the level of the preservatives used in these experiments (Wattenberg 1972) is much higher (5-10 mg/g of feed) than what would be added to animal feed to prolong its shelf life and will therefore probably not result in any significant inhibition of carcinogenesis. 7.5.3 Inconsistencies in EPA and FDA Tolerance Limits for Contaminants A couple of inconsistencies apparently exist between the EPA and FDA tolerance limits for aflatoxin and DDT. The EPA Good Laboratory Practice Standards (USEPA 1979) specifies a maximum limitation of 5 ppb for aflatoxin (Bi, 82, GI, and 62) in animal feed, whereas the Food and Drug Administration Compliance Guide (USFDA 1980b) recommends legal action if the aflatoxin level in the finished feed ingredients is above 20 ppb. The EPA Good Laboratory Practice Standards (USEPA 1979) also specifies a maximum limitation of 100 ppb for DDT (total) in animal feed, whereas the FDA (USFDA 1980a) revised as of April 1, 1980, states: The following tolerance is established for residues of DDT resulting from use of DDT as a pesticide on the growing agricultural crop: 100 parts per million in or on dried tomato pomace to be used in dog and cat food at lev- els up to 5 percent by weight of the prepared food. If the residues of IDE (DDD) on tomatoes are also present, the total of both such chlorinated compounds shall not exceed 100 parts per million. > This calculates to be 5 ppm of DDT (total) in the prepared feed, a level 50-fold higher than the limit (100 ppb) proposed in the EPA Good Laboratory Practice Standards (USEPA 1979). This is, however, in con- trast with an action level of 0.5 ppm for residues of DDT, DDE, and TDE individually or in combination in processed animal feed prescribed in the FDA Compliance Policy Guide 7126.27, Attachment D (USFDA 1981). 44 image: ------- 8. MONITORING AND ANALYSIS OF CONTAMINANTS IN FEED To insist that the industry analyze each batch of manufactured feed for the potential contaminants identified in Sect. 7 will put an almost unbearable and expensive analytical burden on them. On the other hand, investigators sometimes assume that because control animals receive the same basic diet as experimental animals, variations in nutrients or con- taminants will not have significant effect on the experimental outcome. Such an assumption may not always be correct since dietary variables can have significant interactions with experimental variables or parameters. The necessity of monitoring the contaminants in animal diets has been addressed in an important paper by Greenman et al. (1980). These authors analyzed commercial rodent feed for a series of nutrients and potential contaminants during a 5-year period. Frequently, Se was found at concen- trations 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. Besides the seasonal and regional variation in the contaminant level of the feed crop, there may also be variation from year to year as revealed in this study. For instance, the DDT concentration in the commercial animal feed was highest in 1975 (152 ppb), whereas it was between 10-18 ppb in 1974 and in 1976-78. These considerations suggest that periodic monitoring of the contaminants in the feed ingredients and the finished feed by the manufacturer and the investigator are necessary to provide adequate quality assurance as specified in USEPA (1979). Current awareness of the farming practice and the environmental regulations should provide clues to the manufacturer as to the nature of the contaminants for which he should analyze. For example, after DDT was banned, its concentration in the animal diet has gone down, and with increasing use of unleaded gaso- line in cars, the concentration of Pb in the environment has been reduced, with consequent reduction of the level of Pb in human blood (Sterba 1982). Similar reduction in the Pb level in food crops can prob- ably be expected. As previously mentioned, there is no point in setting tolerance limits for contaminants at a level below the analytical capability for detection and assay of the contaminants. In many cases the detection limits and accuracy of analysis are unknown. The development of new analytical methods and refinement of the existing methods for assay of contaminants at ppm/ppb levels should continue to be pursued by the scientific community. The variability in the results of the assay methods widely adopted in commercial and research laboratories should be investigated by collaborative studies as described in AOAC (1980). While no effort was made to include a comprehensive coverage of the literature for different analytical methods for contaminant analysis, the methods in use at the NIH laboratories are briefly described in Appendix A, and pertinent information concerning the analytical methods currently in use at Ralston Purina Company Laboratories at St. Louis is given in Table 19. 45 image: ------- Contaminant Pb Cd Hg Se As Aldrin, Heptachlor, Dieldrin, Matrix Alfalfa Cereal Feed Feed Feed Feed Feed Average recovery 86.7% 107.4% 93.2% Mean level of analyte 2.98 ppm 1.41 ppm 0.46S ppm 131 ppb 1 ppm 0.03% Standard deviation 0.29 ppm 0.14 ppm 0.032 ppm 8 ppb 0.12 ppm 0.002% Coefficient of variation 9.76% 9.65% 6.89% 6.4% 11% 5% Confidence level/ sample wt 0.1 ppm/10 g O.OS ppm/10 g, 0.02 ppm/25 g SOppb/l.Og O.OS ppm/ 1.0 g 0.2 ppm/ 1.0 g 0.02 ppm/10 g References for analytical methodology a b c d e f DDE, DDT, Lindane, Chlordane, Endrin, BHC, HCB, Minx, Methoxychlor PCB Thimet, Diazinon, Disulfoton, Methyl Parathkn, Malathion, Parathion, Thiodan, Ethion, Trithion Aflatorin Feed Feed Feed 0.15 ppm/10 g 0.02 ppm/10 g 20 ppm/50 g "AOAC (1980), 13th ed, 25.068-25.073. *AOAC (1980), 13th ed., 2.109-2.113, 7.096-7.098, 33.089-33.094; Perkin-Elmer, Analytical Methods for Absorption Spectrophotometry, 3/73. Thorpe, Determination of Mercury in Food Products, Michigan Department of Agriculture (1970), modified. AOAC (1975) 12th ed., 3.078; Watltinson, J.H., AnaL Chem. 38(\). Fluorometric Determination of Se in Biological Material with 2,3-Diaminonaphthalene.; Haddad, P.R. and L-E-^mythe, Talanta, 24, 859, 1974. 'Monior, Williams G.W., Trace Elements in Foods, p. 213, John Wiley and Sons, Inc., NY (1949); Bnmford, F., Poisons (1951) 3rd ed., p. 73, The Blakeston Co., Philadelphia, Pa.; Sandell, E.B., Colorimetric Determination of Traces of Metals, p. 142, Intersrience Publishers, New York, N.Y., 1974. 'Pesticide Analytical Manual, VoL I, Sec. 211.13-211.17, 212.101-212.136; AOAC (1975), 12th ed, 29.001-29.018. f Pesticide Analytical Manual, VoL I & II; AOAC (1975), 12th ed, 29.001-29.018. "AOAC (1975), 12th ed. Supplement Method 26.A01-26.A08. Source: Ralston Purina Company (1982). 46 image: ------- 9. INTERACTION OF DIETARY COMPONENTS Certain components in the laboratory animal diet are known to interact with each other. Awareness of this aspect of animal nutrition is essential for the successful formulation of animal diets. The inter- play among Zn, Se, As, vitamins, amino acids, fatty acids, and phytate is described below, as is the importance of Ca:P and K:Na ratios in the animal diet and the enzyme induction by dietary constituents and contaminants. 9.1 Interplay Among Zn. Se. As. Vitamins, Amino Acids, Fatty Acids, and Phytate Zn plays a role in the metabolism of vitamin A (Smith et al. 1976), and Zn deficiency causes alteration in fatty acid patterns in the skin of rats under essential fatty acid deficiency conditions (Betteger et al. 1979). Phytate present in soy protein interferes with the absorp- tion of Zn; therefore, when soy protein is used as the main source of protein in animal diets the Zn level is usually increased. This is the rationale for the higher Zn content of the AIN-76™ diet of 30 ppm (AIN 1977) compared with the NAS-NRC recommendation of 12 ppm for the rat diet (NAS 1978). Selenium possesses anticarcinogenic properties at low levels in animals and humans (Schrauzer 1977). The uptake of Se can be diminished by Zn, and diets rich in sulfur-containing amino acids diminish the physiological activity or availability of Se (Schrauzer 1977). Arsenic behaves as an antagonist to Se in that it increases the urinary excre- tion of Se in rats and causes a deficiency of Se (Schrauzer 1977). There is also an interrelationship between Se and vitamin E. Supple- ments of Se can prevent or cure liver necrosis in the rat and ozidative diathesis in the chick, which are manifestations of vitamin E defi- ciency. Selenium, however, is ineffective against other manifestations of vitamin E deficiency such as testicular and embryonic degeneration in the rat and encephalomalacia in the chick. Sulfur-containing amino acids have a sparing effect on the vitamin E requirement in certain cir- cumstances and offer at least partial protection against liver necrosis and ozidative diathesis (Clark et al. 1977). 9.2 Ca;P Ratio The Ca:P ratio is important in animal nutrition and should be main- tained between 1.0 and 1.5 (NAS 1978). As it has been pointed out by one commenter (comments on the proposed EPA guidelines), this ratio is more important than the absolute amounts of Ca and P in the diet. This fact should possibly be emphasized in the EPA GLP (USEPA 1979). 9.3 K:Na Ratio The K:Na ratio varies from a low of 1.69 in Purina canine diet #5007 to as high as 7.20 in the NAS-NRC recommended diet for the rat (Tables 1. 2, respectively). The protective effect of K in rats during high Na intake in the diet has been reported (Heneely 1973). The role 47 image: ------- of the K:Na ratio in animal diets should be investigated particularly in view of the recent controversy of the value of a low sodium diet in reducing hypertension (Eolata 1982). 9.4 Enzyme Induction and Inhibition of Neoplasia by Minor Dietary Constituents, Feed Additives, and Contaminants A few representative examples of interaction between dietary con- stituents and toxicants have been given in Table 17 and discussed briefly in Sect. 7.5.1. In this section the focus is on enzyme induc- tion and its role in inhibition of neoplasia. 9.4.1 Enzyme Induction by Dietary Constituents Many xenobiotics are capable of inducing microsomal enzymes. These indueers are of at least two types, exemplified by phenobartital (P-450) and 3-methylcholanthrene (P-448) (Conney 1967). The microsomal mixed- function oxidase system is a complex biochemical entity that metabolizes a wide variety of xenobiotics including chemical carcinogens. Three components of the system have been identified: cytochrome P-450, cyto- chrome P-450 reductase, and a lipid. Two distinctive cytochromes have been identified in liver microsomes: naturally occurring P-450 and a closely related species, P-448 (also called Pi~450). Considerable work has been done on one of the components of the cytochrome P-450 system, aryl hydrocarbon hydroxylase (AHH) activity, which is induced by many chemicals and dietary factors. A basal level of AHH activity is present in the liver but not in the small intestine or lungs. AHH activity and also glntathione sulfur-transferase activity was shown to be induced in these tissues by natural ingredient diets such as Purina Rat Chow (Wat- tenberg 1982). Later investigations revealed that most of the inducing activity was associated with the vegetable component, which consisted of alfalfa meal. Naturally occurring inducers were found in cruciferous plants such as brussels sprouts, cabbages, and cauliflower and identi- fied as indole derivatives. Flavones naturally occurring in citrus fruits, safrole, iso-safrole, and 0—ionone have also been found to be enzyme inducers (Wattenberg 1975). 9.4.2 Enzyme Induction by Feed Additives Preservatives, butylated hydroxyanisole and ethoxyquin, have been shown to be good enzyme inducers and consequently reduce the carcino- genic effect of chemicals such as benzo(a)pyrene, 7,12-dimethyl- benzanthracene, and dimethylnitrosaniline (Wattenberg 1979) (Table 16), However, in these experiments, much higher levels of preservatives (5-10 mg/g) were used than is normally present in animal feed (8-20 ug/g). 9.4.31 Enzyme Induction by Contaminants in Animal Feed Data on enzyme induction by pesticide residues in animal feed are rather scarce. Chlordane, DDT, hexachlorocyclohexane, dieldrin, aldrin, heptachlorepoxide, and pyrethrums have been reported to be inducers of enzyme activity (Conney 1967) and, therefore, if present in animal diets, have the potential to alter the outcome of long-term toxicity 48 image: ------- experiments by inducing enzymes that may alter the metabolism of the chemical under study. 9.4.4 Inhibition of Neoplasia Food contains a large number of inhibitors of carcinogenesis such as phenols, indoles, aromatic isothiocyanates, methylated flavones, cou- marins, plant sterols, selenium salts, protease ihibitors, ascorbic acid, tocophorols, retinol, and carotenes. At present, the mechanism of inhibition of carcinogenesis by these factors is poorly understood. An attempt has been made to classify these inhibitors into three different classes according to the time in the carcinogenic process that they are effective. The compounds that prevent the formation of ultimate carci- nogenesis from precursors belong to the first group (e.g., o-tocopherol, ascorbic acid). In the second group are compounds that inhibit carcino- genesis by preventing carcinogenic agents from reaching or reacting with critical target sites in the tissues (so-called blocking agents). The third category of inhibitors acts subsequent to exposures to carcinogens (so-called "suppressive agents"). Of these three categories, the second category or the "blocking agents" most often act as indncers of enzymes (Wattenberg 1983). 9.4.5 Monitoring of Induced Enzyme Activity The most frequently monitored induced enzyme activities are aryl hydrocarbon hydroxylase (Wiebel et al. 1974) and glutathione S-transferase activity (Sparnins et al. 1982). UDP-glucuronyl- transferase, epoxide hydratase (Wattenberg 1980). aminopyrene demethy- lase, p-nitroanisole demethylase, acid phosphatase, and glucose phospha- tase (Newberne 1982) have also been monitored. 9.4.6 Enzyme Induction and Genetic Differences Induction of cytochrome Pi~450 (also called P-448) by polycyclic aromatic compounds has been manifested in the B6 mouse (the inbred C57BL/6 strain) and in other responsive inbred mouse strains, but induc- tion of this form of cytochrome by polycyclic aromatic hydrocarbons is absent in the liver and markedly decreased in all other tissues examined in the D2 (the inbred DBA/2 mouse strain) and other nonresponsive inbred mouse strains. This responsiveness to aromatic hydrocarbons has been termed the Ah locus: the allele Ah^ denotes the B6, and Ahd the D2 inbred mouse (Nebert et al. 1977). It has been shown that when mice are given massive intraperitoneal doses of benzo(a)pyrene, the survival*time of the three responsive inbred strains - B6, C3H/HeN, and BALB/cAmN - is significantly shorter than that of the two nonresponsive inbred strains, D2 and AHR/N. In contrast, when benzo(a)pyrene is administered orally in smaller daily doses, the nonresponsive mouse survives less than 4 weeks, whereas no significant earlier death rate is seen in genetically responsive mice, even after ingesting benzo(a)pyrene for 6 months. Similar differences have been observed when benzo(a)pyrene was substituted by arochlor 1254, lindane, or hexachlorobenzene. Eepone has also been shown to induce AHH activity in B6 but not in D2 mice (Nebert et al. 1977). 49 image: ------- 10. SPECIAL TOPICS Phytates, arsenic, and selenium are treated separately in this sec- tion due to the special concern about them in recent years. 10.1 Phytates Oberleas (1973) provides a comprehensive discussion of phytates as a naturally occurring toxicant in food. Of all the ingredients used in animal feeds, probably soybean meal and wheat are the richest sources of phytate. The phytic acid from plant sources has been identified to be myoinositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate). Consideration of phytates in animal nutrition is important because they can interfere with the absorption of metal ions from the gut. At pH 7.4 phytates form complexes with metals in the following decreasing order: Cu^+ > Zn2+ > Co2* > Mn2+ > Fe3+ > Ca2+. An important factor in the precipitation of metal phytates is the synergistic effect produced when two or more metal ions are present simultaneously; by acting together they increase the quantity of metal phytate precipitated. This phenomenon has been demon- strated for Zn and Ca and for Cu and Ca. Oberleas states that when diets contain plant materials (e.g., soybean) rather than casein as a major source of protein, it is advisable to increase the amount of Zn intake. For this reason the AIN-76^* purified rat and mouse diet uses 2.5 times the level of Zn recommended by NAS (see Tables 1 and 2, respectively). Calcium absorption in the gut is influenced by phytate, vitamin D, and other dietary factors. Diets that have enough Ca, P, and vitamin D and calcium: inorganic phosphorus ratios between 1:1 and 2:1, respec- tively, are not likely to be rachitogenic even though much calcium may be bound to the phytates. 10.2 Arsenic The role of arsenic (As) in the animal diet is controversial (Shen- driker 1982, Huff 1982, Frost 1982). Although there is inadequate evi- dence for the carcinogenicity of arsenic compounds in animals, there is sufficient evidence that inorganic arsenic compounds are skin and lung carcinogens in humans (IARC 1980). Recently Frost (1982) has focused on the noncarcinogenicity of As and on its anticarcinogenicity. High As intake has led to reduced spontaneous lung cancer in mice (Kanisawa and Schroeder 1967, 1969), and epidemiological data indicate a correlation between low As intake and high lung cancer rate (Schrauzer 1978). Niel- sen et al. (1975) have shown that the offspring of Sprague-Dawley dams placed on a purified diet containing approximately 30 ppb As, and main- tained in a plastic isolator environment, displayed a rough coat and a significantly slower rate of growth than controls receiving a supplement of 4.5 ppm As as sodium arsenate (4 ppm) and arsenite (0.5 ppm). At 12- 15 weeks the deficient males exhibited significantly low hematocrits and enlarged and blackened spleens containing 50% more iron on a per gram basis. The beneficial effects of As on the growth, health, and feed efficiency of pigs and poultry have been well established (Frost 1967). 50 image: ------- 4-Nitrophenylarsenic acid and 3-nitro—4-hydroxyphenylarsonic acid have been found to be effective growth stimulants in pigs and poultry, and phenylarsenoxides are more potent than arsenic acids as coccidiostats. Arsenic can act as an antagonist to selenium (Se) and has been used suc- cessfully to alleviate Se poisoning in animals (Underwood 1977). Whether As should be treated as a micronutrient like Se in animal nutri- tion is still an open question. Unfortunately, no information document- ing safe levels for long-term feeding studies is available. 10.3 Selenium The role of selenium (Se) as an essential micronutrient in animal feeds has been well established. It has been shown to be a constituent of glutathione peroxidase, an enzyme involved in the disposal of perox- ides in body tissues. Selenium and vitamin E supplement each other in most animal species studied except in rabbits where the relationship does not hold (MAS 1977). Superiority of natural Se over selenite Se in increasing the Se levels of muscle and liver has been demonstrated in laying hens (Latshaw 1975). Biological availability of Se was shown to be only 54-58% as great from tuna as from selenite for induction of glntathione peroxidase in liver and in red blood cells of rats (Douglass et al. 1981). The Se in most of the feed stock of plant origin was highly available, ranging from 60-90%, but was less than 25% available when the feed stock was of animal origin. This assay was based on the prevention of exudative diathesis induced in chicks on a Se-deficient diet (Cantor et al. 1975). Although Se is an essential micronutrient, an overdose causes res- triction in food intake in rats and dogs together with anemia and severe pathological changes in the liver (Moxon and Rhian 1964). Biliary Se excretion is increased when subacute doses of As are administered. Arsenic has been used as an antidote to Se poisoning, and mercury, copper, and cadmium are also known to interact with Se and offer protec- tion against Se poisoning (Underwood 1977). Contrary to earlier experi- ments, Se has been demonstrated to have an anticarcinogenic effect (Weisberger and Suhrland 1956). Selenium (sodium selenite, ~0.5 mg/week) is undergoing clinical trial for the treatment of endemic car- diomyopathy in China (Sadler 1982). Selenium requirements in animal feeds are usually met by the selenium naturally occurring in feed stock. However, when naturally insufficient, it may be necessary to add Se to the animal feed. At present selenium is recognized as a carcinogen, and its incorporation into commercially prepared laboratory animal feed is subject to the Delaney anticancer clause in the Federal Food, Drug, and Cosmetic Act; but in view of the fact that Se is an essential micronutrient in labora- tory animal diets, the Food and Drug Administration has decided not to enforce this regulation in this particular case. The legal position of the Food and Drug Administration is explained by Ballitch (see Appendix C). 51 image: ------- 11. ANALYSIS OF PUBLIC COMMENTS ON THE PROPOSED EPA GUIDELINES FOR DIETARY REQUIREMENTS AND CONTAMINANT ANALYSIS 11.1 Criticisms of Proposed Guidelines and Related Discussion Several commenters agreed with the EPA that a standardized diet should be required and should meet minimal requirements because dietary constituents can influence the incidence of disease, including neo- plasms, and certain dietary contaminants may be linked to synergistic, additive or antagonistic effects in animal studies. Many commenters, however, disagreed with the EPA approach on several grounds: (1) feasibility, (2) practicality, (3) scientific justification, (4) lack of harmony with FDA, USDA, etc., (5) cost effectiveness, (6) lack of incentive to continue research on diet development, and (7) failure to promote standardization of the diet. The changes sug- gested in the EPA approach include (1) deletion of Appendix A and B from the proposed guidelines and (2) adoption of a different set of criteria for diet standards. The focus of EPA's concern in these proposed guidelines is long- term tozicity testing of chemicals using laboratory animals. The fact that dietary constituents and contaminants can affect the outcome of the tests is well recognized. The customary use of a set of control animals for the purpose of comparing with the exposed animals is not an adequate safeguard since dietary variables can have significant interactions with experimental variables or parameters (Greenman et al. 1980). These con- siderations justify the EPA approach to set the guidelines for the dietary requirements and contaminant analysis. However, most labora- tories procure their animal feed from commercial sources. A major share of the burden of complying with the EPA regulations will thus fall on the laboratory animal feed manufacturers. As testified by several com- menters, the industry has established a good record of adequate service to the needs of the scientific community. The commenters feel that the EPA regulations should be flexible and permit the investigator and the feed manufacturer a greater latitude in the choice of feed ingredients, use of preservatives, and analytical requirements. As an example of the difficulty that feed manufacturers have in ensuring the use of proper ingredients, consider alfalfa, which used to be oven-dried but now is mostly field-dried for economic reasons with consequent reduction in quality of the resulting feed. It is customary to analyze the vitamins and minerals in the premixes and to analyze one or two components in the finished product to ensure proper distribution. Vitamins that are partially destroyed dur- ing the. manufacturing process, such as vitamin C, have to be assayed in the finished product. Some of the analysis may be costly, time- consuming and unnecessary if carried out routinely; for instance estro- genic activity is usually not assayed unless it is relevant to the par- ticular experiment. The analytical burden on the industry should be kept at a minimum without compromising the objectives of the EPA Good Laboratory Practice. It should be noted that as is evident from the 52 image: ------- data presented in Table 1, the animal feed industry is already meeting many of the standards set by the EPA. 11.2 Analysis of Specific Comments 11.2.1 Shelf Life Restrictions Numerous commenters argued that the restriction on shelf life (90 days) is unwarranted. Consideration should be given for the use of preservatives such as ethozyquin and BHA, except where these compounds are known to interact with the chemical under investigation. In fact, as shown in Table 8, NIH diet formulations do use preservatives. If preservatives are allowed, the shelf life of most laboratory feeds is accepted to be about 6 months except in case of guinea pig feed where the shelf life is usually 3 months because of rapid deterioration of vitamin C. However, if supplemented with vitamin C, shelf life of guinea pig feed can also be extended to 6 months. These shelf life data are valid only when the feed is kept at 70°F or lower and a relative humidity of 50% under clean storage conditions (personal communication from D.C. SheIton of Ralston Purina 1982). 11.2.2 Feed Meal Justification of the requirement that the feed in meal form should be manufactured by regrinding pellets has been questioned. Pelletized feed for long-term studies has been suggested. Joseph Knapka of NIH has retracted his statement (Knapka 1979) that volatile nitrosamine levels in animal feed are substantially lowered during the pelletizing process (personal communication from J. Knapka 1982); therefore, there apparently is little justification for preparing the feed in meal form by regrinding the pellets, although it may be preferable to do so to minimize biological contaminants and to subject the feed to the same heat treatment as the pellets. Whether autoclaving reduces nitrosamine concentrations in the feed, as suggested by one commenter, needs to be documented. 11.2.3 Calcium, Phosphorus, Iron, and Cobalt The EPA proposed minimum for calcium (Ca) and phosphorus (P) for rodent diets are 1.15% and 0.9%, respectively. Calcium and phosphorus in some commercial rodent diets range from 0.9% to 1.1% and from 0.6% to 0.85%, respectively (see Table 1). One commenter stated that the EPA minimum levels for Ca and P are rather high. According to NAS (1978) the Ca:P ratio in the diet is more important than their absolute percen- tage in the feed and should be greater than one. Consideration should be given to lowering the proposed minimum levels of Ca and P in the diet and also for including a specified minimum for the Ca:P ratio. The EPA proposed minimum for iron in rodent diets is 345 ppm. Lev- els of iron in some commercial rodent diets range from 180 ppm to 298.7 ppm (see Table 1). The NAS recommendation for iron in rodent diets ranges from 25 ppm to 140 ppm (see Table 2). As has been pointed out by 53 image: ------- one commenter, the minimum level of iron in the proposed EPA guidelines seems rather high, and consideration should be given to lowering the level of iron. One commenter stated that cobalt is not a requirement under normal animal husbandry. This agrees with the NAS recommendation for the rat diet. However, cobalt is a requirement for the golden hamster; the amount required in the diet has been estimated to be 1.1 ppm (see Table 2) (NAS 1978). It is required for the synthesis of vitamin Bj.2, and all commercial rodent feed formulations include cobalt, 0.38 ppm to 0.96 ppm (Table 1), to ensure proper nutrition. 11.2.4 Vitamins The minimum level of vitamin E proposed in the EPA guidelines is 3 ppm. One commenter has pointed out that the vitamin K level of 3 ppm is totally inadequate for normal blood clotting in certain widely used strains of rodents. The most satisfactory level appears to be 15 ppm according to this commenter. Rodents meet a large part of the dietary need for vitamin K through intestinal synthesis and coprophagy (NAS 1978). Purina rodent chow #5002, guinea pig chow #5026, and rabbit chow #5322 do not include vitamin K (Table 1); however, the Agway R-M-H 3000 diet does include 0.97 ppm of vitamin K (Table 1). These facts favor the retention of the proposed level; however, the requirement for higher levels in certain strains of rodents should be further investigated. One commenter questioned the absence of vitamin C in the proposed guide- lines. Vitamin C is required in guinea pig diet formulations (NAS 1978). Other animals do not require a dietary source of vitamin C, and commercial diet formulations for the mouse, rat, dog, hamster, and rab- bit do not include vitamin C (Table 1). Since the guinea pig is a rodent the EPA guidelines should include vitamin C if they are intended for all rodents. As pointed out by one commenter, alternative sources of thiamine (thiamine hydrochloride) and niacin (niacinamide) should be considered for inclusion in the proposed EPA guidelines to allow flexibility in diet formulations. A question was raised on whether EPA should allow the use of vita- min D—activated vegetable sterol along with vitamin D-activated animal sterol. Vitamin D-activated animal sterol is a source of vitamin DS, and vitamin D-activated vegetable sterol is a source of vitamin D2 (AAFCO 1982). Although no information was available for rodents, vita- min DS is preferred over D2 for poultry diets (Merck Index 1976). The commercial diet formulations listed in Table 1 use vitamin D-activated animal sterol. No data are readily available to suggest that the use of vitamin D-activated vegetable sterol be permitted. 11.2.5 Choline and Inositol One commenter observed that the minimum level of choline proposed in the EPA guidelines (USEPA 1979) is too high (1900 ppm). The guaranteed minimum level of choline in rodent diets varies from 1542 ppm 54 image: ------- to 2000 ppm in commercial diets (Table 1). The NAS recommendation for choline in the diet of different animals ranges from a low of 600 ppm (mouse) to a high of 2000 ppm (golden hamster) (Table 2). No reason is readily apparent for changing the proposed EPA value unless it is desired to provide values for individual species. One commenter questioned the absence of inositol in the EPA guide- lines. Inositol is not normally required in the diet of laboratory animals. None of the commercial diet formulations include inositol (see Table 1). However, the NAS recommended diet for the golden hamster does require 100 ppm of inositol (NAS 1978) (see Table 2). 11.2.6 Alternative Sources of Minerals One commenter pointed out that the permitted sources of minerals include manganese oxide and cobalt carbonate but do not include man- ganese sulfate nor cobalt sulfate. Since the two latter compounds are included in the GRAS list (see Table 7), their use in the EPA guidelines should be considered to allow flexibility in the diet formulation. 11.2.7 Dicalcium Phosphate One commenter expressed concern about the potential effects of fluorine usually associated with dicalcinm phosphate, which is specified as a source of minerals in the proposed guidelines. Specifications for diealelorn phosphate include a maximum permissible level of fluorine of no more than 1 part to 100 parts of phosphorus (AAFCO 1982). Most com- mercial diet formulations do not state the amount of fluoride present; the Agway R-M-H 3000 diet is an exception and contains an average of 35 ppm of fluoride (see Table 1). Consideration should be given for speci- fying an acceptable level of fluoride in the diet. 11.2.8 Fat The minimum percentage of fat allowed in the proposed EPA guide- lines is 4.3%. One commenter remarked that in the case of adult, nonbreeding rodents, the use of a lower percentage (2-3%) of fat in the diet avoids obesity and perhaps shortening of life span. Commercial rodent diets include 4-5% fat (see Table 1). The NAS recommendation for the rodent diet also recommends inclusion of 5% fat (see Table 2). 11.2.9 Ash One commenter stated that the minimum ash content of 8% in the EPA guidelines is rather excessive, particularly for adult rodents. The maximum ash content of some commercial rodent feeds is guaranteed to be 6% (Agway Inc. n.d.) or 7% (Ralston Purina Company 1980). The specified ash content in the EPA guidelines possibly should be revised accord- ingly. 55 image: ------- 11.2.10 Air Dry One commenter asked fox a definition of "air dry" because the EPA guidelines specify that the nutrient content of both ingredients and the finished product must be expressed as a nutrient content percentage by weight on an air-dry basis. Since normal storage conditions of animal feed specify a temperature of 70°F and a relative humidity of 50%, "air dry" feed may be defined as the feed dried to a constant weight under these conditions. Alternatives are to report the results on a dry weight basis or 90% dry matter basis as reported in NAS (1974). 11.2.11 Units of Measurement For the sake of uniformity, consideration should be given to using ppm instead of mg/kg. Vitamins A and D are usually expressed in I.U./g. 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Ann Clin Lab Sci 4:441-447. Reviewed in Greenman et al. 1980. Schranzer GN. 1977. Trace elements, nutrition and cancer: perspectives of prevention. In: Inorganic and nutritional aspects of cancer. Schrauzer GN, ed. New Tork: Plenum, pp. 323-344. Schrauzer GN, White DA, Schneider CJ. 1977. Cancer mortality correlation studies - IV: Association with dietary intakes and blood levels of cer- tain trace elements, notably Se-antagonists. Bioinorganic Chem 7:35-56. Schroeder HA, Balassa JJ. 1967. Arsenic, germanium, tin and vanadium in mice: Effects on growth, survival and tissue levels. J Nutr. 92:245-252. Reviewed in Greenman et al. 1980. 65 image: ------- Schroeder HA, Balassa JJ, Vinton WE Jr. 1964. Chromium, lead, cadmium, nickel and titanium in mice: Effects on mortality, tumors and tissue levels. J Nutr 83:239-250. Reviewed in Greenman et al. 1980. Schroeder HA, Balassa JJ, Vinton WH Jr. 1965. Chromium, cadmium and lead in rats: Effects on life span, tumors and tissue levels. 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Azaserine carcinogenesis: organ susceptibility change in rats fed a diet devoid of choline. Int J Cancer 22:36-39. Silinskas KC, Okey AB. 1975. Protection by l,l,l-trichloro-2,2-bis(p- chlorophenyl)ethane (DDT) against mammary tumors and leukemia during prolonged feeding of 7,12-dimethylbenze(a)anthracene to female rats. J Natl Cancer Inst 55:653-657. Reviewed in Greenman et al. 1980. Silverman J, Adams JD. 1983. N-nitrosamines in laboratory animal feed and bedding. Lab Animal Sci 33(2):161-164. Silverstone H, Tannenbaum A. 1951. Proportion of dietary protein and the formation of spontaneous hepatomas in the mouse. Cancer Res 11:442-446. Smith JC, Brown ED, KcDaniel EG, Chan W. 1976. Alterations in vitamin A metabolism during Zn deficiency and food and growth restriction. J Nutr 106:569. Smith DH, Rogers AE, Herndon BJ, Newberne PK. 1975. Vitamin A (Retinyl Acetate) and Benzo(a)pyrene-indnced respiratory tract carcinogenesis in hamsters fed a commercial diet. Cancer Res 35:11-16. 66 image: ------- Smith DM, Rogers AE, Newberne PM. 1975. Vitamin A and benzo(a)pyrene carcinogenesis in the respiratory tract of hamsters fed a semisynthetic diet. Cancer Res 35:1485-1488. Smith 1C, McDaniel EG, Fan FF, Halsted JA. 1973. Zinc: a trace element essential in vitamin A metabolism. Science 181:954. Sparnins VL, Venegas PL, Wattenberg LW. 1982. Glutathione S-transferase activity: enhancement by compounds inhibiting chemical carcinogens!s and by dietary constituents. J Nat Cancer Inst 68:493-495. Sterba IP. 1982. National study finds lead levels in blood have declined sharply. The New York Times, March 20, p. 8. Stob M. 1973. Estrogens in Foods. In: Toxicants occurring naturally in foods, 2nd ed.. National Academy of Sciences, Washington, DC. Stubblefield RD, Shotwell OL, Hesseltine CW, Smith ML, Hall HE. 1967. Production of aflatozin on wheat and oats: Measurement with a recording densitometer. Appl Microbiol 15:186-190. 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Vol. 1, p. 392, Marcel Dekker, NT. 69 image: ------- APPENDIX A. NATIONAL INSTITUTES OF HEALTH STANDARD FOR NUTRIENT AND CHEMICAL CONTAMINANT ANALYSES OF LABORATORY ANIMAL DIETS NIH SID No. 5A November 1, 1980 Superseding NIH STD No. 5 November 1, 1978 1. Scope 1.1 This specification covers nutrient and chemical contaminant analyses on samples of laboratory animal diets purchased under NIH contracts. 2. Requirements 2.1 Laboratory analyses to be performed a. Nutrient Analyses - Diet samples submitted shall be subjected to the following nutrient analyses in accordance with the most recent issue of Official Methods of Analyses of the Association of Official Analytical Chemists (A.O.A.C.). (1) Moisture (2) Crude Protein (3) Crude Fat (4) Ash (5) Crude Fiber (6) Nitrogen-Free-Extract (7) Calcium (8) Phosphorus Nutrient analyses shall be conducted on duplicate samples. b. Contaminant Analyses - Diet samples submitted shall be subjected to the following contaminant analyses in accordance with the indicated procedure. Contaminant Analysis Method (1) Chlorinated hydrocarbon pesticides Shall be conducted in (2) Polychlorinated biphenyls (PCB's). accordance with A.O.A.C. (3) Organo-phosphate pesticides. section 29.011 for sample preparations. Electron capture detection by gas liquid chromatography. (4) Lead Shall be conducted in accordance with A.O.A.C. section 25.068 for sample preparation; analysis by atomic absorption. 71 image: ------- Contaminant (5) Arsenic (6) Cadmium (7) Mercury Analysis Method Shall be conducted in accordance with Anal. Chem. 48: Page 120, 1976 for sample preparations (discussion on automation may be disregarded). Analysis by atomic absorption. Shall be conducted in accordance with A.O.A.C. section 25.105 for sample preparation; analysis by atomic absorption using a double beam instrument with simultaneous back- ground correction. Shall be conducted in accordance with A.O.A.C. section 25.105 for sample preparation; analysis by atomic absorption. Shall be conducted in accordance with A.O.A.C. section 16.A01-26.A08 (mini column method for combined Bl, B2, Gl, 62). Shall be conducted in accordance with J.A.O.A.C., 51, P. 763, 1968. Contaminant analyses on duplicate samples is not required. Samples to be analyzed under this Specification will be submitted by the NIB animal feed contractors and/or the project officer. The feed manufacturers and the Independent Laboratory performing the analyses will be notified in writing, at the time the contract is awarded, of the diet samples which they are authorized to submit for analyses. The Government will be responsible to pay the cost of analyzing only these authorized samples. 2.2 Frequency of Assays - All specified nutrient and contaminant ana- lyses shall be completed within 28 days after the feed samples are received by the laboratory. The laboratory shall maintain a log indicat- ing the date each sample is received, the name of the company submitting the sample and the complete identification. (Feed contractors are required to identify all samples with the name of the diet, the date of manufacture and the NIH stock number.) (8) Aflatoxins (9) Nitrate 72 image: ------- APPENDIX B. CONVERSION FACTORS FOR SELECTED VITAMINS al IU of Vitamin A = 1 USP unit = 0.3 (ig all-trans retinol = 0.344 fig all-trans retinyl acetate or 0.550 |ig all-trans retinyl palmitate = 0.6 jig of all-trans 0-carotene al mg of 0-carotene = 1667 IU of Vitamin A = 833 IU of Vitamin A activity for the dog bl IU of Vitamin D = 0.025 fig of cholecalciferol bl IU of Vitamin E = 1 mg of dl-o-tocopheryl acetate = 0.91 mg of dl-o-tocopherol bl mg of thiamine HC1 = 0.89 mg of thiamine bl mg of calcium pantothenate = 0.92 mg of pantothenic acid bl mg of pyridozine HC1 = 0.82 mg of pyridozine bl mg of Vitamin ~K~ — 0.73 mg menadione sodium bisulfite or 0.38 mg menadione Source: aNAS (1974). bHerck Indez (1968). 73 image: ------- APPENDIX C. LETTER FROM EDWARD J. BALLITCH April 8, 1982 Dr. B. C. Pal Oak Ridge National Laboratory Post Office Box z Oak Ridge, Tennessee 37830 Dear Dr. Pal This responds further to your telephone call of March 23, 1982, to Dr. Gerald Guest, Acting Bureau Director, and our telephone conversation of March 32, 1982, asking whether the Delaney anticancer clause in the Federal Food, Drug and Cosmetic Act (Act) is applicable to selenium incorporated into commercially prepared laboratory animal feed. This clause, in section 409(c)(3)(A) of the Act (21 U.S.C. 348(c)(3)(A)), prohibits issuance of a food additive regulation for a substance which induces cancer, except that it does not apply with respect to an ingredient of feed for food producing animals under specified conditions. Therefore, under a literal reading of the law, an article which is a car- cinogen may not be approved for use in laboratory animal feed, as you know, selenium has been demonstrated to induce cancer in laboratory animals. The agency recognizes, however, that selenium is an essential nutritional element for laboratory animals and that feed is the most practical method of providing this nutrient. Furthermore, its use for this purpose is very limited and carefully monitored in laboratory animal diets. We have, therefore, concluded that our current position is that we do not intend to take regulatory action against the marketing and use of appropriate supplemental sources of selenium at generally accepted nutri- tional levels in laboratory animal feeds solely on the basis that a regu- lation for this use is prohibited under the Act. Of course, if future information raises safety concerns regarding this policy, the agency will take appropriate measures. Undoubtedly you are aware of data suggesting that trace levels of selenium act to inhibit carcinomas in laboratory animals. We caution that this fact be taken into account when designing toxicity studies involving animals fed selenium in their feed. Selenium levels in labora- tory animal feed should be reported in the study results. Edward J. Ballitch Director Division of Compliance Bureau of Veterinary Medicine Food and Drug Administration Rockville. MD 20857 74 image: ------- APPENDIX D. ABBREVIATIONS LIST AIN American Institute of Nutrition BHA Butylated hydroxyanisole IU International units NAS National Academy of Sciences NDMA N-Nitrosodimethylamine NIB National Institutes of Health NRC National Research Council TON Total Digestible Nutrients USDA United States Department of Agriculture USP United States Pharmacopoeia 75 image: ------- APPENDIX E. ADDITIONAL LITERATURE REFERENCES Aaron A, Schad JS. 1954. Nitrogen balance studies with dogs on casein or methionine supplemented casein. J Nutr 53:265. Ackerman CJ. 1957. Reversal of sulfaguanidine toxicity in the rat. J Nutr 63:131-42. Afonsky D. 1954. Folic acid deficiency in the dog. Science 120:803. Agar J. 1971. Effect of a low zinc diet during gestation on reproduction in the rabbit. J Anim Sci 33:1255. Ahlborg UG, Dich J. 1978. The use of food additives in Sweden. Arch Tox- icol Suppl 1:305-308. American Association for Cancer Research. 1981. Workshop on fat and cancer. Cancer Res 41(9):3677-3826, part 2 of 2. American Institute of Nutrition Ad Hoc Committee Report on Standards for Nutritional Studies. 1977. J Nutr 107(7):1340-1348. Anonymous. 1978. BRIEFING: Nitrosamines found in NIH-approved animal feed. Science 202:192. Arnich L, Lewis EH, Morgan AF. 1952. Growth of dogs on purified diet plus aureomycin and/or vitamin Bl2« Proc Soc Exp Biol Hed 80:401. Arrington LR, Taylor RN Jr, Ammerman CB, Shirley, RL. 1965. Effects of excess dietary iodine upon rabbits, hamsters, rats and swine. J Nutr 87:394. ;' Asburn LL. 1940. The effects of administration of pantothenic acid on the histopathology of the filtrate factor deficiency state in rats. Pub- lic Health Rep 55:1337. Baggs RB, Miller SA. 1973. Nutritional iron deficiency as a determinant of host resistance in the rat. J Nutr 103:1554. Balazs T, Sekella R, Pauls JF. 1971. Renal concentration test in beagle dogs. Lab Anim Sci 21:546. Balk MW, Foster HL. 1982. Histocompatibility and isoenzyme differences in commercially supplied BAL/c mice: A reply. Science 217:381. Ballitch EX. 1982. Personal communication. Letter dated 4/8/82 to Dr. B.C. Pal. Barki VH, Derse PH, Collins RA, Hart EB, Elvehjem CA. 1949. The influ- ence of coprophagy on the biotin and folic acid requirements of the rat. J Nutr 37:443. 76 image: ------- Barnes LL, Sperling 6, Maynard LA. 1941. Bone development in the albino rat on a low manganese diet. Proc Soc Ezp Biol Med 46:562. Barnes RH, Bates MI, Maack J. 1946. The growth and maintenance utiliza- tion of dietary protein. J Nntr 32:535. Barnes RH, Fiala 6. 1959. Effects of the prevention of coprophagy in the rat. VI. Vitamin K. J Nutr 68:603. Barnes RH, Lundberg, WO, Hansen HT, Burr GO. 1943. The effect of certain dietary ingredients on the keeping quality of body fat. J Biol Chem 149:313. Barney GH, Hacapinlac HP, Pearson WN, Dalby WJ. 1967. Parakeratosis of the tongue-a unique pathologic lesion in the zinc deficient squirrel monkey. J Nntr 93:511. Barstad. 1978. Pesticides and heavy metals as food contaminants. Arch Tozicol Suppl 1:47-54. Baur LS, Filer LJ, Jr. 1958. Evaluation of lysine supplementation of infant formulas fed to rats. J Nutr 66:545-54. Beard HH. 1929. Studies in the nutrition of the white mouse. V. The experimental production of rickets in mice. Am J Physiol 89:54. Beaton JR, Beare JL, McHenry EW. 1952. Factors affecting the development acrodynia in pyridozine-deficient rats. J Nutr 48:325. Beck EH, Fenton PF, Cowgill GR. 1950. The nutrition of the mouse. IX. Studies on pyridozide and thiouracil. Yale J Biol Med 23:190. Behne, D, Hoer T, von Berswordt-Wallrabe R, Elger W. 1982. Selenium in the testis of the rat: studies on its regulation and its importance for the organism. J Nutr 112:1682-1687. Bender AD, Schottelium DD, Schottelius BA. 1959. Effect of short-term vitamin E deficiency on guinea pigs, skeletal muscle myoglobin. Am J Physiol 197:491. Bennett MJ, Coon E. 1966. Mellituria and post-prandial blood sugar curves in dogs after the ingestion of various carbohydrates with the diet. J Nntr 80:163-168. Berg BN, Simms HS. 1961. Nutrition and longevity in the rat. III. Food restriction beyond 800 days. J Nutr 74:23. Bergman EN, Sellers AF. 1960. Comparison of fasting ketosis in pregnant and nonpregnant guinea pigs. Am J Physiol 198:1083. Bessey OA, Wolbach SB. 1939. Vascularization of the cornea of the rat and riboflavin deficiency with a note on corneal vascularization and vitamin A deficiency. J Ezp Med 69:1. 77 image: ------- Best CH, Huntsman HE, Solandt OM. 1932. A preliminary report on the effect of choline on fat deposition in species other than the white rat. Trans R Soc Can 26:175. Best CH, Huntsman HE. 1932. The effects of the component of lecithin upon deposition of fat in the liver. J Physiol 75:405. Best CH, Hershey JM, Huntsman ME. 1932. Effect of lecithin on fat depo- sition in the liver of the normal rat. J Physiol 75:56. Bethke RM, Kick CH, Wilder W. 1932. The effect of calcium-phosphorus relationship on growth, calcification, and blood composition of the rat. J Biol Chem 98:389. Bild CE. 1959. Raising healthy pups. Proc Animal Care Panel 9:79-82. Bloodworth 1MB. 1965. Experimental diabetic glomerulosclerosis. II. The dog. Arch Pathol 79:113. Bloom WL, Azar GJ. 1963. Similarities of carbohydrate deficiency and fasting. Arch Intern Med 112:333. Boas MA. 1927. An observation on the value of egg white as a sole source of nitrogen for young growing rats. The effect of dessication upon the nutritive properties of egg white. Biochem J 21:712. Boelter MDD, Greenberg DM. 1943. Effect of severe calcium deficiency on pregnancy and lactation in the rat. J Nntr 26:105. Boffey PM. 1982. In wake of mouse mix-up, studies are left in ruin assessing the effects of mixnp in lab mice. The NY Times, July 27, 1982. Borchers R. 1958. Effect of dietary level of raw soybean oil meal on the ground of weanling rats. J Nutr 66:229-233. Bosshardt DE, Paul WJ, Barnes RH. 1950. The influence of diet composi- tion on vitamin Bl2 activity in mice. J Nntr 40:595. Bouman J, Slater EC. 1956. Tocopherol content of heart muscle prepara- tions. Nature 177:1181. Bourne GH. 1957. Some aspects of the feeding of dogs and other car- nivora. Laboratory Animals Bureau, MRC Laboratories, London. Collected papers 5:81-87. Braham JE, Villarreala A, Bressani R. 1962. Effect of lime treatment of corn on the availability of niacin for cats. J Nntr 76:183. Bras G, Ross MH. 1964. Kidney disease and nutrition in the rat. Toxicol Appl Pharmacol 6:247. Brody JE. 1982. A sizeable number of Americans may not get enough zinc. The NY Times; July 28, 1982. 78 image: ------- Brown D, Ferguson JD, Ramsay AG. 1953. Guinea-pigs reared on a diet con- taining synthetic ascorbic acid. I Physiol (London) 121:36-37P. Brown EB, Smith DE, Dubach R, Moore CB. 1959. Lethal iron overload in dogs. J Lab Clin Med 53:591. Browning CB, Parrish DB, Fountaine FC. 1958. Effect of feeding low lev- els of diethylstilbestrol on gestation and lactation of rats. J Nutr 66:321-322. Bryan WL, Mason KE. 1940. Vitamin E deficiency in the mouse. Am J Phy- siol 131:263. Bryan GT, Brown RR, Price JM. 1964. Incidence of mouse bladder tumors following implantation of paraffin pellets containing certain tryptophan metabolites. Cancer Res 24:582. Buckley GP, Hartroft WS. 1955. Pathology of choline deficiency in the mouse-observations with special reference to liver. Arch Pathol 59:185. Bull LS, Hurley WL, Kennett WS, Tamp1in CB, Williams WF. 1974. Effect of sex hormones on feed intake in rats. J Nutr 104:968. Bunce GE, Jenkins ES, Phillips PH. 1962. The mineral requirements of the dog. III. The magnesium requirement. J Nutr 76:17. Burr GO, Burr MM. 1930. On the nature and role of the fatty acids essen- tial in nutrition. J Biol Chem 86:587. Campbell HL. 1945. Seasonal changes in food consumption and rate of growth of the albino rat. Am J Physiol 143:428-433. Campbell JE, Phillips PH. 1953. Some problems on feeding fat to dogs. Southwest Vet 6:173. Campbell TC. 1981. A decision tree approach to the regulation of food chemicals associated with irreversible tozicities. Regulatory Toxicology and Pharmacology 1:193-201. Cantor AH, Longevin ML, Noguchi T, Scott ML. 1975. Efficacy of selenium in selenium compounds and feedstuffs for prevention of pancreatic fibrosis in chicks. J Nutr 105:106-111. Cantor AH, Scott ML. Noguchi T. 1975. Biological availability of selenium in feedstuffs and selenium compounds for prevention of exuda- tive diathesis in chicks. I Nutr 105:96-105. Carpenter KT, Kodicek E. 1948. The blood picture in pyridoxin and riboflavin deficiencies in rats. Br J Nutr 2:ix. Carroll KE, Ehor HT. 1971. Effects of level and type of dietary fat on incidence of mammary tumors induced in female Sprague-Dawley rats by 7,12-dimethylbenz(a)anthracene. Lipids 6:415. 79 image: ------- Cassidy J. 1957. The commercial manufacture of compressed diets for laboratory animals. Laboratory Animals Bureau, MRC Laboratories, London. Collected Papers 5:15-19. Chapin RE, Smith SE. 1967. Calcium requirement of growing rabbits. J Anim Sci 26:67. Chesney AM, Clawson TA, Webster B. 1928. Endemic goitre in rabbits. 1. Incidence and characteristics. Bull John Hopkins Hosp 32:261. Christensen F, Dam H. 1953. A new symptom of fat deficiency in hamsters: profuse secretion of cerumen. Acta Physiol Scand 27:204. Cohen ML, Reyes P, Typpo JT, Briggs GM. 1967. Vitamin Bl2 deficiency in the golden hamster. J Nutr 91:482. Cohez. ML, Arnrich L, Okey R. 1963. Pantothenic acid deficiency in .ixolesterol-fed hamsters. J Nutr 80:142. Cole W, Earned BK. 1938. Diabetic traits in a strain of rats. Endocri- nology 23:318. Committee on Animal Nutrition. 1966. Nutrient requirements of domestic animals. Biological Energy Interrelationships and Glossary of Energy lu^rr* let icvised edition 1966. Cooperman JM, Waisimen HA, Elvehjem CA. 1943. Nutrition of the hamster. Proc Soc Ezp Biol Med 52:250-254. Coots MC, Harper AE, Elvehjem CA. 1959. Production of biotin deficiency in the guinea pig. J Nutr 67:525. Corbin J. 1976. Laboratory animal nutrition. In: CRC handbook on labora- tory animal science. Melby GC Jr, Actman NH, eds. Vol. 3. Cordes DO, Mosher AH. 1966. Brown pigmentation (lipofuscinosis) of the canine intestinal muscularis. J Pathol Bacteriol 92:197. Cotes PM, Reid E, Toung FG. 1949. 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Performing Organization Name and Address Information Center Complex P.O. Box X Oak Ridge National Laboratory Oak Ridge, TN 37831 10. Project/Task/Work Unit No. 11. Contract(C) or Grant(G) No. (C) (G) DW930141-01-1 12. Sponsoring Organization Name and Address Office of Toxic Substances U.S. Environmental Protection Agency 401 M Street SW Washington, DC 20460 13. Type of Report & Period Covered Final 14. 15. Supplementary Notes This report supersedes PB&4111541. 16. Abstract (Limit 200 words) The primary objectives of this report are to present information concerning the nutritional requirements of several commonly used laboratory animal species (i.e., mouse, rat, hamster, guinea pig, rabbit, and dog) and to discuss various aspects of the problem of contamination of laboratory animal feeds. In addition, this document discusses the different types of laboratory animal diets (e.g., open vs closed formula), the ingredi- ents used in these diets, the interaction of dietary components, and the public comments received respective to the EPA proposed guidelines for the nutrient composition of laboratory animal diets. Much of the data are presented in tabular form. 17. Document Analysis •. Descriptors Laboratory animals Diets Nutrition Vitamins b. Identifiers/Open-Ended Terms Preservatives Contamination Impurities Pesticides Nitroso compounds Estrogens Fungi Molds Aspergillus Fusarium c. COSATI Field/Group Biology 18. Availability Statement Release unlimited 19. Security Class (This Report) Unclassified 20. Security Class (This Page) Unclassified 21. No. of Pages 118 22. Price (SeeANSI-Z39.18) See Instructions on Reverie 101 OPTIONAL FORM 272 (4-77) (Formerly NT1S-35) Department of Commerce image: -------