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
-------
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thereof.
-------
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
-------
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
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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
-------
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
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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
-------
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
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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.
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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
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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
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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).
-------
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.
-------
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
-------
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.
-------
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.
-------
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
-------
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).
-------
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.)
-------
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.)
-------
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
-------
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.).
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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
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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
-------
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 ^
-------
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
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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
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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
-------
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
-------
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
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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).
-------
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
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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
-------
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
-------
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
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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
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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
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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.
-------
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
-------
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
-------
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).
-------
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
-------
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
-------
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
-------
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
-------
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)
-------
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.
-------
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
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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.
-------
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
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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
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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
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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
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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
-------
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
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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
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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
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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
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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
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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
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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
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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.
The use of units such as Mcg/lb should be avoided, as indicated by one
commenter.
56
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•»
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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.
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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
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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
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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
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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
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5*0272-101
REPORT DOCUMENTATION
PAGE -
1. REPORT NO.
EPA 560/6-83-005
3. Recipient's Accession No.
4. Title and Subtitle
Nutritional Requirements and Contaminant Analysis of
Laboratory Animal Feeds
5. Report Date
May 1984
7. Authors)
-Bimal C. Pal, Robert H. Ross, Harry C. Milman (EPA)
8. Performing Organization Rept. No.
ORNL
9. 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
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