Protein Efficiency Ratio (PER) Rat 
Bioassay Studies to Demonstrate that a 
New Infant Formula Supports the 
Quality Factor of Sufficient Biological 
Quality of Protein: Guidance for 
Industry 
Draft Guidance 
This guidance is distributed for comment purposes only. 
Although you can comment on any guidance at any time (see 21 CFR 10.115(g)(5)), to ensure 
that we consider your comment on this draft guidance before we begin work on the final version 
of the guidance, submit either electronic or written comments on the draft guidance within 90 
days of publication in the Federal Register of the notice announcing the availability of the draft 
guidance.  Submit electronic comments to https://www.regulations.gov.  Submit written 
comments to the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 
Fishers Lane, rm. 1061, Rockville, MD 20852.  All comments should be identified with the 
docket number FDA-2022-D-2424 listed in the notice of availability that publishes in the 
Federal Register. 
For questions regarding this draft document, contact the Center for Food Safety and Applied 
Nutrition (CFSAN) at 240-402-1450. 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Food Safety and Applied Nutrition 
February 2023  Contains Nonbinding Recommendations 
Draft-Not for Implementation 
Table of Contents 
I. Introduction ....................................................................................................3 
II. Background ....................................................................................................4 
III. Overview of AOAC Official Method 960.48  Protein Efficiency Ratio
(PER), Rat Bioassay ......................................................................................6 
IV. Appropriate Modifications of AOAC Official Method 960.48 .............. 8
A. Need for Appropriate Modifications to Update the AOAC Method
and for Use of Infant Formulas in PER Bioassays ........................................ 8
B. Conduct and Analysis of a PER Study with Appropriate
Modifications (matching the casein reference (control) and test diets) ...... 9
V. Miscellaneous ...............................................................................................2  9
A. Protocols and Reports ........................................................................2  9
B. Reference Guidelines .........................................................................3  1
VI. References .....................................................................................................3  2
VII. Appendices ....................................................................................................3  5
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Protein Efficiency Ratio (PER) Rat 
Bioassay Studies to Demonstrate that a 
New Infant Formula Supports the 
Quality Factor of Sufficient Biological 
Quality of Protein: Guidance for 
1
Industry  
This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
Administration (FDA or we) on this topic.  It does not establish any rights for any person and is 
not binding on FDA or the public.  You can use an alternative approach if it satisfies the 
requirements of the applicable statutes and regulations.  To discuss an alternative approach, 
contact the FDA staff responsible for this guidance at the phone number listed on the title page. 
I. Introduction
The Infant Formula Act of 1980 (Pub. L. 96-359) amended the Federal Food, Drug, and 
Cosmetic Act (FD&C Act) to include section 412 (21 U.S.C. 350a).  In 1986, Congress amended 
section 412 of the FD&C Act to require FDA to establish quality factors for infant formula and 
stipulated that an infant formula would be considered adulterated if it does not meet the quality 
factor requirements.2  On June 10, 2014, as part of the final rule, Current Good Manufacturing 
Practices, Quality Control Procedures, Quality Factors, Notification Requirements, and Records 
and Reports, for Infant Formula (Infant Formula Final Rule), FDA established requirements for 
quality factors for infant formulas (79 FR 33057), including the quality factor of sufficient 
biological quality of protein (21 CFR 106.96(e) and (f)). 
An infant formula must meet the quality factor of sufficient biological quality of protein (21 CFR 
106.96(e)).  Specifically, 21 CFR 106.96(f) describes how an infant formula manufacturer must 
demonstrate that a formula meets this quality factor: 
1 This guidance has been prepared by the Office of Nutrition and Labeling, Infant Formula and Medical Foods Staff 
intheCenter for FoodSafetyandAppliedNutritionincooperationwiththeOfficeofRegulationsandPolicyatthe 
U.S. Food and Drug Administration.  
2 See Anti-Drug Abuse Act of 1986, Pub. L. 99-570,  4014. 
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A manufacturer of an infant formula that is not an eligible infant formula shall demonstrate 
that a formula meets the quality factor of sufficient biological quality of protein by 
establishing the biological quality of the protein in the infant formula when fed as the sole 
source of nutrition using an appropriate modification of the Protein Efficiency Ratio (PER) 
rat bioassay described in the Official Methods of Analysis of AOAC International, 18th 
ed., sections 45.3.04 and 45.3.05, AOAC Official Method 960.48 Protein Efficiency Ratio 
Rat Bioassay, which is incorporated by reference at  106.160 (see Appendix 1).  The PER 
rat bioassay shall be conducted on a formula and the results evaluated prior to the initiation 
of a growth monitoring study of the formula that is required under 21 CFR 106.96(b). 
Conducting a PER study in an animal model3 permits a determination of a formulas protein 
quality before infants are exposed to the formula.4  This approach ensures that infants will not be 
fed a formula with inadequate or biologically unavailable protein.5 
We have developed this guidance to help manufacturers and laboratories in the design, conduct, 
evaluation, and reporting of PER studies.  The guidance is intended to explain how the PER 
study can be used to provide assurance that a new infant formula meets the quality factor of 
sufficient biological quality of protein in the infant formula when fed as the sole source of 
nutrition using appropriate modifications of AOAC Official Method 960.48 (the AOAC Method; 
Reference 1) (see 21 CFR 106.96(f)). 
In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
as recommendations, unless specific regulatory or statutory requirements are cited. The use of 
the word should in Agency guidances means that something is suggested or recommended, but 
not required. 
II. Background
The purpose of the PER methodology, as defined in the AOAC Method, is to determine the 
efficiency (i.e., quality) of a test protein ingredient compared with that of the reference casein 
protein from a rat bioassay.  The quality is determined by comparing growth rates from rats 
consuming a diet containing the test protein ingredient with those of rats consuming a diet 
containing the reference casein protein.  The two diets should be similar in composition to avoid 
the possibility of creating confounding variables.  The PER method defines the dietary 
3 We support the principles of the 3Rs to reduce, refine and replace animal use in testing when feasible.  We 
encourage sponsors to consult with us if they wish to use a non-animal testing method they believe is suitable, 
adequate, and validated to demonstrate that the formula supports the quality factor for the biological quality of the 
protein as described in 21 CFR 106.96(g)(3).  We support alternative methods by exemption in 21 CFR 106.96(f) 
which allows the manufacturer to request an exemption and provide certain required assurances described in 21 CFR 
106.96(g).  The applicability of this exemption is not the subject of this guidance. 
4 Current Good Manufacturing Practices, Quality Control Procedures, Quality Factors, Notification Requirements, 
and Records and Reports, for Infant Formula, Interim Final Rule, 79 FR 7934, at 8023, Feb. 10, 2014. 
5 79 FR at 8023. 
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components in the protein evaluation basal diet and adjustments needed to ensure that all 
comparisons between the test sample and the reference casein are made with diets having a 
similar content of nitrogen, fat, ash (i.e., inorganic residual remaining after water and organic 
matter have been removed by heating), moisture, and crude fiber. 
Despite recognized limitations, the PER bioassay is unique in its ability to assess relative protein 
utilization.  Chemical measurements of total protein (i.e., measurement of nitrogen) and 
determination of amino acid patterns are also possible and may be appropriate for certain aspects 
of protein quality determinations (Reference 2).  However, such chemical measurements do not 
address the bioavailability of a protein.  The PER study permits a comparison of the 
bioavailability of different protein sources.  We are not aware of any other method to assess 
protein bioavailability.  In addition, the PER studys standardization as the AOAC Method has 
led to improved reproducibility (Reference 3). 
FDA regulations require that the protein in an infant formula be evaluated as part of the whole 
formula matrix, rather than as a single protein ingredient, to account for the effects of processing 
or other components on digestion and protein availability.6  One challenge with this requirement 
is how to control for components in the formula matrix that fall outside of the original 
description of the bioassay in the AOAC Method (References 4 through 7).   
It is well-recognized that the composition of certain food products can influence the assessment 
of the PER (References 4, 8).  Modifications to the diets described in the AOAC Method are 
necessary because infant formulas have relatively high fat and low protein contents and may 
contain high concentrations of lactose.  The aim of specific diet modifications is to ensure that 
the infant formula containing the test diet and the casein reference diet are as similar as possible 
so that protein quality can be evaluated without being confounded by other differences between 
the test and reference diets.  Adjustments to diet compositions from those described in the 
AOAC Method are made to attain similar levels and types of fat, carbohydrate, and ash content.  
In addition, an infant formula brings its own complement of vitamins and minerals into the PER 
study test diets.  The matching  of these additional nutrients in the PER study reference and test 
diets is challenging.  Without proper modifications to the reference diets from what is specified 
in the AOAC Method, the PER values of infant formulas may be underestimated in comparison 
to casein (Reference 7). 
Modifications to the diets described in the AOAC Method are also necessary to bring the AOAC 
Method into alignment with current knowledge of rat nutrition.  Since the development of the 
AOAC Method in the 1960s, there have been significant developments in understanding the 
nutrient requirements of rats.  With respect to infant formula, FDA considers modifications to the 
diet described in the AOAC Method that are based on updated knowledge of rat nutrition to be 
appropriate modifications of the AOAC Method within the meaning of 21 CFR 106.96(f). 
6 See 21 CFR 106.96(f). 
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Section III provides information about the AOAC Method as it was originally written. 
Section IV provides recommendations that address appropriate modifications to update the 
AOAC Method to bring it in line with current knowledge of rat nutrition, as well as 
recommendations that address appropriate modifications of the AOAC Method for infant 
formula. 
III. Overview of AOAC Official Method 960.48  Protein
Efficiency Ratio (PER), Rat Bioassay
The AOAC Method was developed in the early 1960s and provides a procedure by which the 
quality of a protein can be evaluated and compared with those of other proteins.  Protein 
quality can be defined as the ability of a protein to meet the essential amino acid needs of an 
animal, including humans.  The AOAC Method is a standardized bioassay, and its collaborative 
study data have been published7 (References 9, 10). 
The AOAC Method permits the calculation of a PER as the ratio between the average animal 
body weight gain per gram of protein consumed of a test protein versus casein after a 28-day 
feeding period.  Typically, to improve the sensitivity of the method, the protein concentration of 
both the test and reference diets is set at about 10%, a level that is below the estimated level 
required for growth of 15% (References 3, 11).  While growth is slower at 10% protein than at 
15% protein, the lower protein levels ensure that available protein is used efficiently. 
PER studies need to be carried out under standardized conditions that include age and species of 
rat, diet composition including protein levels, and duration of feeding and feeding method, so 
that studies with matrices containing different proteins can be compared (Reference 12).  These 
conditions are defined in the AOAC Method.  Certain conditions of the PER study are not 
defined (e.g., strain of rat, diet fed during acclimation period), while others are defined within 
specified ranges (e.g., age of rats, length of acclimation period, number of rats per group, 
frequency of measurements). 
While the AOAC Method is flexible in many respects, ensuring consistency between the test and 
control groups with regard to diet composition is critical to ensure the assay is specific for 
protein quality and the comparison is not influenced by other uncontrolled differences between 
the test and reference diets.  To ensure the test and reference diets are matched except for protein 
source, adjustments are needed to the reference diet specified in the AOAC Method.  In 
7 Two multi-laboratory collaborative studies provided validation data for the AOAC Method.  PER values for the 
casein control groups were (grams (g) body weight gain/g protein consumed for the 28-day study period):  2.79  
0.34 (Reference 9 sucrose as carbohydrate source), 3.12  0.22 (Reference 10 starch as carbohydrate source), and 
2.66  0.26 (Reference 10) sucrose as carbohydrate source).  FDA considers PER values between 2.4 and 3.3 to be 
reasonable for casein control groups under the original conditions of the assay. 
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conducting a PER assay, the performance of the contemporaneous reference group8 is as critical 
to the evaluation of the results as the performance of the test group because the results are 
reported as relative rather than as absolute values (Reference 4). 
The PER study defined in the AOAC Method (see Appendix 1) provides a specific nutrient 
composition and formulas for adjustments that are needed to ensure that the test and reference 
diets have similar contents of nitrogen, fat, ash, moisture, and crude fiber.  General 
compositional requirements and adjustments are summarized below.  An example of the 
formulation for the control diet for a PER study as originally described in the AOAC Method is 
shown in Appendix 2. 
AOAC Method Diet Formulation and Adjustments 
Dietary 
Ingredient  level,  Dietary adjustment 
% 
Protein  ......10  Protein from test sample 
Cottonseed oil           8  Minus fat content of test sample 
Salt mixture           5  Minus ash content of test sample 
Vitamin mixture           1  No adjustment described 
Cellulose           1  Minus fiber content of test sample 
Water           5  Minus moisture content of test sample 
Sucrose or corn starch  To 100 
According to the AOAC method, proximate analysis9 is needed to adjust the diets so that all 
comparisons between the test protein ingredient and the reference casein protein are made with 
diets having similar overall contents of nitrogen, fat, ash, moisture, and crude fiber (Reference 
1).  Thus, the minimum specifications for chemical analyses for PER study diets include the 
determination of nitrogen, fat, ash, moisture, and crude fiber.  Additional analyses can be   
performed if desired, and many methods are available (e.g., Official Methods of Analysis of 
AOAC International, AOAC International, Rockville, MD; Official Methods and Recommended 
Practices of the American Oil Chemists Society, American Oil Chemists Society, Urbana, IL; 
and Approved Methods of Analysis of the Cereals and Grains Association, Cereals and Grains 
Association, St. Paul, MN). 
8 Historical control group data (e.g., data from groups of rats fed casein diets in earlier studies) are generally unable 
to replace a contemporaneous control group because diets used in generating such data are unlikely to match the 
composition of the infant formulation under consideration.  It is also unlikely that details of such historical studies 
including age of rats, duration of study, acclimation period and diet, study diet compositions, etc. would be the same 
as those in the AOAC Method. 
9 Proximate analysis refers to the quantitative analysis of protein, fat, moisture, ash, and crude fiber.  Carbohydrate 
is calculated by difference. 
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Formulas are provided in the AOAC Method to allow calculation of reference diet composition 
based on results of proximate analysis of the test material.  Examples of applications of such 
calculations are shown in Appendix 2. 
IV.  Appropriate Modifications of AOAC Official Method 960.48 
A.  Need for Appropriate Modifications to Update the AOAC Method and for Use of 
Infant Formulas in PER Bioassays 
Since the publication of the AOAC Method in the 1960s, there have been significant 
developments in our understanding of the nutrient requirements of rats.  Thus, for PER studies 
generally, there is a need for updates to the AOAC Method to ensure such studies are conducted 
in a manner consistent with the most current understanding of nutrient requirements of rats.  
With respect to infant formula PER studies, FDA considers changes to the AOAC Method that 
are based on such current understanding of nutrient requirements of rats to be appropriate 
modifications of the AOAC Method under 21 CFR 106.96(f). 
There is also a need for appropriate modifications of the AOAC Method when determining the 
PER of infant formulas.  In the PER study as originally described in the AOAC Method, a 
protein ingredient was assayed at 10%, and other potential variables (e.g., age of rats, diet 
compositions, length of study) were standardized to minimize confounding variables (Appendix 
1).  Vitamin composition, moisture, ash, carbohydrates, fat, and fiber were similar for both 
reference and test diets.  Use of a test diet that includes an infant formula in its entirety 
introduces matrices of high fat content and additional vitamins, minerals, and other ingredients 
as well as low protein content.  A major challenge in analyzing infant formulas by the AOAC 
Method is matching the reference and test diets to achieve dietary groups with as few 
confounding variables as possible. 
Studies that have evaluated variables that might be encountered when determining the PER of 
infant formulas have found that modifications of the levels and sources of fat and carbohydrate 
in casein reference diets to match those in various infant formulas led to significant changes in 
the resultant PER study values (Reference 7).  For example, PER values (g body weight gain/g 
protein consumed for the 28-day study period; mean  SEM10; 10 rats/group) varied from 3.2  
0.13 in an unmatched casein reference diet to 2.3  0.24 and 2.1  0.16 in two matched, milk-
based formula test diets (Reference 7).  The PER value of 2.1 is 66% of the value measured in 
the unmatched reference group and shows the magnitude of the difference that may result when 
reference and test diets are not well-matched.  In these data, the PER values of the milk-based 
infant formulas appear to be significantly underestimated in comparison to the PER value of the 
casein reference group.  The protein quality evaluation of infant formulas using the PER 
 
10 Standard Error of the Mean (SEM) is an estimate of how far the sample mean is likely to be from the population 
mean. 
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bioassay warrants the use of matched casein reference diets for each type of formula (Reference 
7). 
B.  Conduct and Analysis of a PER Study with Appropriate Modifications (matching 
the casein reference (control) and test diets) 
1.  Preparation of Experimental Diets 
Limitations with respect to the assessment of the quality of protein sources for infant formulas 
using the AOAC Method can be greatly reduced by appropriate modifications of the test and 
reference diets.  Generally, studies with groups of rats fed unmatched diets are very difficult to 
interpret because the results are confounded by too many variables.  For this reason, the 
compositions of the reference and test diets should be followed throughout the manufacturing 
process and adjustments to procedures made, as needed, to avoid such confounding variables.  
The composition of the reference diet in a PER study is within the scope of the investigators 
responsibility, and many adjustments can be made in order to match the composition of the 
casein reference diet to that of the infant formula-based test diet.  In the sections below, 
references to the casein control or control or casein reference control refer to the casein 
reference diet. 
As stated in FDAs interim final rule, Current Good Manufacturing Practices, Quality Control 
Procedures, Quality Factors, Notification Requirements, and Records and Reports, for Infant 
Formula (Infant Formula Interim Final Rule): 
Prior to study initiation, the test product (finished infant formula) and the casein control are 
subjected to a compositional assessment (proximate analysis).  The diets are then formulated 
to contain matching amounts of protein, fat, minerals, fiber, and moisture.  These diets are 
analyzed for protein to confirm that they were formulated correctly, which information is 
used to calculate the PER at completion of the trial. 
79 FR 7934, 8023 (Feb. 10, 2014).11  The Infant Formula Interim Final Rule also stated, 
Although the method has limitations with respect to assessment of the quality of protein sources 
for infant formulas, the limitations are greatly reduced by modification of the test and control 
diets (79 FR 7934 at 8023) and provided details on [t]hree dietary adjustments commonly 
required for evaluation of the protein quality of infant formulas: (1) matching of the fat content 
of the reference and test diets; (2) matching of the carbohydrate composition of the reference and 
test diets; and (3) removal of water from liquid infant formula to achieve the lower limit of 
nitrogen (1.8% by weight) specified by the PER bioassay (79 FR 7934 at 8023 to 8024).  FDA 
also has considered other areas in which modifications may be appropriate, including the 
 
11 The preamble to FDAs interim final rule describes the appropriate modifications of the AOAC Method.  The 
Infant Formula Final Rule adopted, with some modifications not applicable here, the Infant Formula Interim Final 
Rule.  See 79 FR 33057. 
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matching of minerals and vitamins in the reference and test diets, acclimation diets and length of 
acclimation, and record-keeping. 
In addition, as noted above, FDA also has considered areas in which modifications may be 
appropriate to update the AOAC Method to reflect current knowledge of nutrient requirements of 
rats. 
The sub-sections below describe FDAs current thinking with respect to preparation of PER 
study casein reference diets; protein source, conversion of nitrogen to protein; fats and 
carbohydrates (including a discussion of vitamin E as it relates to content of polyunsaturated 
fatty acids (PUFA)); removal of water from liquid infant formulas and determination of moisture 
in PER study diets; mineral content; vitamin content; fiber; sulfur amino acids (SAA) 
(methionine, cystine); other constituents (including vitamin C); developing estimates of 
compositions of PER study reference and test diets (minerals and vitamins); and chemical 
analysis (demonstrating the appropriateness of modifications). 
a.  Preparation of PER study casein reference diets 
Preparation of PER study casein control diets that are matched as closely as possible to the test 
diet poses several challenges because of the high fat and high lactose contents of many infant 
formulas.  Mixing of the ingredients may result in a reference diet of gummy or pasty 
consistency, which may be unacceptable to the young animals.  Such consistency also may lead 
to problems with poor digestibility and reduced nutrient availability, and, as a result, the PER 
study reference diet may lead to very slow or no growth during the 28-day study period.  
Manufacturers and laboratories may want to consider blending or combining the fats, protein, 
carbohydrates, vitamin, and mineral mixtures, and other ingredients using general processes and 
equipment such as that used in the manufacture of infant formulas, if such are available.  Use of 
such procedures may reduce variables related to differences in diet consistency (texture) between 
the casein reference and the infant formula test diets.  Records of how the PER diets are prepared 
should be included in the protocol, maintained with the diet preparation records of the study, and 
included in the final report (see Section V.A., below). 
b.  Protein Source, Conversion of Nitrogen to Protein 
In the AOAC Method, the protein content of a product is calculated from its nitrogen content by 
applying a factor considered suitable for converting nitrogen to protein in the food.  Such factors 
are based on the nitrogen content of the predominating protein present in various foods.  The 
AOAC Method specifies the use of the factor 6.25 as the nitrogen conversion factor for setting 
protein levels. 
The AOAC Method identifies the Animal Nutrition Research Council (ANRC) reference casein 
as the protein source for the reference diet.  Several high-purity caseins ( 85%;  90%) are 
available from commercial suppliers.  Results of amino acid analyses are often available with the 
specifications for these products.  For PER studies generally as well as infant formula PER 
studies specifically, FDA considers these high-purity caseins to be appropriate for use as the 
protein source for the reference diet.  Regardless of the casein product used, we recommend that 
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all specifications for the casein used in PER studies, including the results of the amino acid 
analyses, be retained because it is helpful in confirming the adequacy of the reference diets with 
respect to amino acid composition. 
c.  Fats and Carbohydrates 
Fat content: The original protein evaluation basal diet described in the AOAC Method 
contained 8% cottonseed oil, which was readily available in the past, but is no longer readily 
available.12  With respect to infant formula, FDA stated the following in the Infant Formula 
Interim Final Rule: 
In most cases, when the infant formula is incorporated into the protein evaluation diet based 
on the nitrogen content, the fat content will be above the limit (8 percent) specified by the 
AOAC Official Method.  The fat content of the reference control (casein) diet must be 
adjusted to match the fat content of the infant formula test diet. 
(79 FR 7934 at 8024.) 
Infant formulas contain a variety of fat sources, and FDA considers it appropriate and possible to 
match the fat content and fatty acid compositions of the PER study reference diet both 
quantitatively and qualitatively to that of the infant formula-based test diet.  This matching of fat 
and fatty acid compositions is important because, in both humans and animals, the bodys need 
for vitamin E increases with an increase in consumption of PUFAs and with the degree of 
unsaturation of dietary PUFAs. 
The rat requirement for vitamin E (RRR--tocopherol; formerly called d--tocopherol) is 18.0 
milligrams per kilogram (mg/kg) diet (equivalent to 27 international units (IU)/kg) when lipids 
comprise less than 10% of the diet (e.g., a diet containing 50.0 grams (g) fat/kg, 6.0 g linoleic 
acid (n-6)/kg, and an estimated requirement of 2 g/kg linolenic acid (n-3), which can be 
substituted with other long-chain n-3 PUFA)13 (Reference 11).  Higher concentrations of vitamin 
E may be required if high-fat diets are fed (Reference 11).  Harris and Embree reviewed studies 
in which rats were fed diets containing varying ratios of vitamin E:PUFA (expressed as mg of d-
-tocopherol to grams of PUFA), which were sufficient either to induce vitamin E deficiency or 
to relieve it (Reference 13). 
 
12 Previously, many laboratories used corn oil (Reference 5) or blends of coconut and corn oils or coconut and soy 
oils (References 7, 8) in place of cottonseed oil.  While corn oil is still readily available, many oils are now blends of 
the most economically available vegetable oils (Reference 6).  No consensus seems to have developed in favor of 
the inclusion of a specific oil or oil blend for the 8% fat PER study diets.  However, in the studies cited, the same oil 
or oil blend was used at the same concentration in both PER study reference and test diets. 
13 The terms "n-3" (or omega-3) and "n-6" (or omega-6) identify fatty acids belonging to two series of 
polyunsaturated fatty acids.  The "n-3" and "n-6" refer to where the first double bond occurs in the molecule.  In the 
omega-3 fatty acids, the first double bond occurs on the 3rd carbon atom - counting from the methyl (or omega) end. 
In the omega-6 fatty acids, the first double bond occurs on the 6th carbon atom, again counting from the methyl (or 
omega) end. 
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Harris and Embree (Reference 13) found that across diets varying from 5 to 22% in total fat 
content and including fat sources such as lard, cod liver, linseed oil, corn oil, and menhaden oil, a 
minimum ratio of vitamin E:PUFA of 0.48  0.28 mg of d--tocopherol to grams of PUFA 
(mean  standard deviation; n=9 studies) was needed to protect against vitamin E deficiency. 
While it is not possible to provide a rule for predicting the requirement for a nutrient without 
taking into account other components of the diet, the vitamin E:PUFA ratio may serve as a useful 
guideline.  The following examples illustrate the use of the minimum ratio value for vitamin 
E:PUFA: a diet containing 10% soybean oil with 58% PUFA and a vitamin E content of 1.8 
mg/100 g has a vitamin E:PUFA ratio of 0.31 (1.8/5.8=0.31), which may have an adverse effect 
on vitamin E nutrition.  The ratio can be increased by adding more vitamin E to the diet as 
follows: the same diet containing 10% soybean oil with 58% PUFA and a vitamin E content of 
5.0 mg/100 g has a vitamin E:PUFA ratio of 0.86 (5.0/5.8=0.86). 
We suggest that the total PUFA contents of the PER study test and reference diets be estimated 
from the Certificates of Analysis or other information and used with dietary concentrations of 
vitamin E to calculate the ratio of vitamin E:PUFA as mg vitamin E/g PUFA for each diet.  
Modifications in the concentration of vitamin E in the reference diet may be made, if needed. 
Carbohydrates:  As originally developed, PER study diets were made up to 100% with sucrose 
or corn starch.  Rats can utilize several carbohydrates, including glucose, sucrose, maltose, 
fructose, and a variety of starches (e.g., corn, wheat, rice) (Reference 14).  No consensus seems 
to have developed for use of a specific carbohydrate composition.  As noted in References 6 
through 8, the same type and level of carbohydrates were used in both the PER study reference 
and test diets. 
With respect to infant formula, as FDA stated in the Infant Formula Interim Final Rule: 
Lactose is the carbohydrate component of most milk-based infant formulas.  Rats do not 
tolerate lactose well and often develop diarrhea, which may lead to an underestimate of protein 
quality of the formulas.  The casein reference control diet(s) must contain levels of lactose 
comparable to the amount in the infant formula test diet to adjust for possible confounding of 
the estimation of protein quality.  If an infant formula contains a carbohydrate source other 
than lactose (e.g., sucrose, corn syrup solids), the source of carbohydrate in the formula should 
be used in the control diet as well. 
(79 FR 7934 at 8024.) 
Products high in lactose represent a special category of foods because of the adverse responses of 
rats to diets with lactose (Reference 15).  FDAs current thinking is that it is critical to the 
conduct of a well-designed PER study that the casein reference diet contain a level of lactose 
comparable to amounts present in the infant formula test diet.  The need for inclusion of lactose 
in acclimation diets fed prior to a PER study with a high-lactose test diet is considered below. 
 
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d.  Removal of Water from Liquid Infant Formulas and Determination of 
Moisture in PER Study Diets 
Removal of Water from Liquid Infant Formulas: As stated in the Infant Formula Interim 
Final Rule: 
Infant formula is incorporated into the protein evaluation diet based on its nitrogen content.  
Because of the high water content of infant formulas in liquid form, these products are below 
the limit of total nitrogen (1.8% by weight) required for the PER bioassay.  Liquid infant 
formulas must be freeze dried so that the test sample contains more than 1.8% nitrogen before 
the infant formula test diet is formulated. 
(79 FR 7934 at 8024.) 
Either powdered or liquid forms of an infant formula may be used in a PER study.  Freeze-drying 
(lyophilization) is a reasonable way to accomplish the removal of water from a liquid infant 
formula.  If a powdered form of an infant formula is used in a PER study, the amount used 
should be adjusted so that the test sample contains more than 1.8% nitrogen before the infant 
formula test diet is formulated. 
Determination of Moisture in PER Study Diets: The appropriate modifications that FDA 
discussed in the Infant Formula Interim Final Rule did not address the issue of water in the PER 
diets (a separate issue from the removal of water from liquid infant formulas with the goal of 
bringing the nitrogen level to the concentration specified in the AOAC Method).  Furthermore, 
the issue of the water content of the PER study diets is distinct from the issue of providing 
drinking water to the animals during the study,14 and one cannot substitute for the other.  The 
water content of the diets needs to be considered in formulating the PER study diets because 
adjustment for water (expressed as moisture in the formula shown in the AOAC Method; 
Appendix 1) is one of the primary specifications in the development of the PER study diets.  
PER study reference and test diets should both contain equal moisture contents because the level 
of hydration may influence PER study results (Reference 4).  The AOAC Method provides a 
formula for adjusting the water content of PER diets to about 5% based on the moisture content 
of the test material.  Earlier studies have shown that differences in water content may influence 
PER study results, with PER values for casein increasing with the percent of water added to the 
diets (Reference 4 and references therein).  A second effect of water that should be considered is 
the point at which water is added to the diets.  In earlier studies, higher PER values were 
obtained when water was added to casein at the beginning of the diet mixing process than when 
water was added to the final diet after mixing other ingredients (Reference 15).  FDA 
recommends that the issue of water in PER study diets be discussed with diet formulators with 
experience in blending diet ingredients such as oils and carbohydrates to avoid problems with 
diet consistency or palatability. 
 
14 See Section IV.B.3.a for further information about providing drinking water to animals during the study. 
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e.  Mineral Content 
The AOAC Method is flexible with respect to the mineral mixture used to meet the mineral 
levels defined in the reference and test diet preparations.  The AOAC Method provides the 
composition of a specific United States Pharmacopeia (USP) salt mixture (See Appendix 1) and 
also states that other salt mixtures having essentially the same proportions of all essential 
elements, including sulfur, may be used.  The mineral mixture described in the AOAC Method is 
USP XIX (1975) (p. 612).  The corresponding item in USP XX (1980) is found in USP XX (141) 
at page 902.  Both citations refer to the same salt mixture, which is part of USPs Protein-
Biological Quality Test.  When used at 5% of the diet, this mineral mixture provides the minerals 
shown in Appendix 3. 
In the formulation of the mineral mixture used in the AOAC Method diets, magnesium (Mg), 
iron (Fe), manganese (Mn), Zn, and copper (Cu) are provided as their sulfate salts.  These salts 
provide about 96.4 mg sulfur/100 g to the final diet (Appendix 3). 
The following paragraphs describe FDAs recommendations regarding mineral content, 
specifically: (1) FDAs recommendations regarding the mineral mixture to be used, taking into 
consideration the sulfur/sulfate content of the final diets, with recommended changes in the 
concentrations of specific minerals in the mineral mixture to update the AOAC Method to bring 
it into alignment with current knowledge; and (2) FDAs recommendations regarding ash content 
and evaluating the mineral composition of the final PER study test diet relative to that of the 
reference diet. 
Mineral mixtures:  Except for zinc (Zn), selenium (Se), and molybdenum (Mo), the 
concentrations of minerals provided in the USP salt mixture are within the acceptable range in 
the estimated nutrient requirements for growth of the laboratory rat provided in the National 
Academy of Sciences, National Research Council (NRC) Nutrient Requirements of Laboratory 
Animals (Reference 11).  These estimated nutrient requirements for growth are minimal 
requirements and do not include a margin of safety (Reference 11).  The concentration of iodine 
(I), while not at a potentially toxic level, is about 200-times higher than its concentration in 
currently available mineral mixtures. 
FDA considers the mineral mixture described in the AOAC Method with the following changes 
to be appropriate for use at 5% with the 10% protein PER study diets that are low in methionine 
(Reference 16): (1) the concentration of potassium iodide (KI) should be reduced from 0.79 g/kg 
of mixture to 0.0079 g/kg of mixture; (2) the concentration of zinc sulfate (ZnSO  7H O) should 
4 2
be increased from 0.548 g/kg of mixture to 1.1 g/kg of mixture; (3) sodium selenate (Na SeO ) 
2 4
should be added at 0.0080 g/kg of mixture; (4) ammonium paramolybdate ((NH ) Mo O   
4 6 7 24
4H O) should be added at 0.0056 g/kg of mixture; and (5) cobalt (Co), which is not required by 
2
rats (Reference 11), should be omitted.  When the resultant mineral mixture is used at 5% of the 
diet, the concentrations of I, Zn, Se, and Mo will be (g/kg diet) 0.00030, 0.0125, 0.00017, and 
0.00016, respectively, and will meet the specifications of the NRC Nutrient Requirements of 
Laboratory Animals (Reference 11). 
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We also consider the use of the Bernhart-Tomarelli mineral mixture (Reference 17) to be 
appropriate when 0.010 g Na SeO and 0.0070 g (NH ) Mo O  4H O are added per kg of the 
2  4   4  6  7  24 2
mixture.  When used at 4% of diets, this Se- and Mo-supplemented mixture will provide 0.00017 
g Se/kg diet and 0.00016 g Mo/kg diet and will meet the specifications in the NRCs Nutrient 
Requirements of Laboratory Animals (Reference 11). 
Other mineral mixtures are commercially available from manufacturers of animal feeds.  The 
mixtures include AIN-76A and AIN-93G, which are constituents of, respectively, rodent 
diets AIN-76A and AIN-93G.  We do not recommend the use of the AIN-76A or AIN-93G 
mineral mixtures in the 10% casein PER study diets because of their sulfur/sulfate content. These 
mineral mixtures provide sulfate, as potassium sulfate, at 0.301-0.337 g/kg diet (Appendix 3) 
compared with 0.501 g sulfate/kg from the Bernhart-Tomarelli mineral mixture (used at 4%) and 
0.964 g sulfate/kg from the USP XIX mineral mixture (used at 5%) in the AOAC Method control 
diet.  Use of the AIN-76A or AIN-93G mineral mixtures alone leads to situations in which the 
inorganic sulfur/sulfate contents of the final diets are lower than that of the finished diet 
described in the AOAC Method.  Our current thinking is if a decision is made to use one of the 
AIN mineral mixtures, then the inorganic sulfate concentration of the diets should be adjusted to 
provide the inorganic sulfate content set forth in the AOAC Method (i.e., 0.964 g/kg diet).15  
The composition of each of the above mineral mixtures is listed in Appendix 3. 
Ash content and evaluation of the mineral composition of the diets:  Regardless of which 
mineral mixture is used, FDA recommends a thorough evaluation of the mineral composition of 
the final PER study test diet relative to that of the reference diet.  The AOAC Method as 
originally written specifies the use of a mineral mixture at 5% of the diets, less an adjustment for 
the ash content of the test material.  Our current thinking is that the ash content of diets is not an 
appropriate surrogate for mineral composition when PER studies are conducted with infant 
formulas. 
Infant formulas contain significant quantities of minerals, and the usual adjustment, as specified 
in the AOAC Method, is to add less of a mineral mixture to the test formula diet than is added to 
 
15 Sulfur has not been classified as a required nutrient in experimental rat diets but is an integral part of the sulfur-
amino acids and vitamins (Reference 14).  However, Michels and Smith showed that dietary sulfate is readily 
incorporated into cartilage of adult rats and will spare methionine for this purpose (Reference 18).  They suggested 
that 0.1% dietary sulfur be included when methionine is minimal.  These findings were supported by Bernhart and 
Tomarelli, who reported that a mineral mixture that met the NRC (1963) mineral specifications for rats (which did 
not include sulfur) was improved by the inclusion of 0.1% sulfate when fed in a low-protein diet (8.8% lactalbumin) 
(References 17, 19).  The authors concluded, based on their results and other studies cited in Reference 17, that with 
diets containing suboptimal levels of protein and low levels of sulfate, the addition of sulfate results in increased 
growth and PER.  Therefore, a salt mixture for comparing protein quality should contain enough sulfate to allow for 
variations in the sulfate content of the proteins tested (References 16, 17).  Based on these and several other reports, 
the NRCs Nutrient Requirements of Laboratory Animals (1978) (Reference 14) for rats included a specification for 
sulfur (i.e., 0.03 g/100 g diet; 0.03%; 0.3 g/kg diet).  The NRCs Nutrient Requirements of Laboratory Animals 
(Reference 11) also cited studies reported in Reference 13 and concluded that 300 mg sulfur/kg diet (0.3 g/kg diet) 
as inorganic sulfate may be beneficial in diets containing 15% protein. 
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the casein reference diet based on ash content (Appendix 1, Appendix 2).  The magnitude of this 
adjustment is much greater when infant formula is used as the test material than when an isolated 
protein source is used as the test material.  For example, a PER study reference diet formulated 
to contain 5% of a mineral mixture and 10% casein with an ash content of 1.51% would contain 
4.85% of the mineral mixture (5.0%  (10%)(1.51)/100 or 5.0% - 0.15% = 4.85%).  The 
corresponding PER study test diet formulated with 84.25% of an infant formula with an ash 
content of 3.83% would contain only 1.77% of the same mineral mixture (5.0%  
(84.25%)(3.83)/100 or 5.0%  3.23% = 1.77%).  The resultant ash contents of both finished diets 
will be the same (i.e., 5.0 % in the example above).  However, calculations of amounts of 
individual minerals added by the infant formula and those added by the mineral mixture will 
likely show that the overall mineral compositions of the PER study test and reference diets are 
not comparable because the relative proportions of minerals added to the test diet by the infant 
formula plus the mineral mixture differ significantly from those added to the reference diet by 
the mineral mixture alone. 
Thus, while our current thinking is that the ash content alone is not an appropriate surrogate 
when matching minerals in test and reference diets in a PER study with infant formulas, we  
recommend that this issue be resolved by developing estimates of compositions of projected PER 
study test and reference diets prior to initiation of the PER study.  In Section IV.B.1.j. and 
Appendix 6, FDA provides a discussion and a table that shows how the compositions of PER 
study test and reference diets can be developed from the composition of the infant formula. 
f.  Vitamin Content 
The AOAC Method provides the composition of a vitamin mixture and specifies its use at 1% in 
both the PER study reference and test diets.  This specification indicates that the expectation of 
the AOAC Method is that both reference and test diets will have the same vitamin compositions, 
thus reducing the confounding effects of having different vitamin compositions between the 
reference and test groups. 
The vitamin mixture as listed in the AOAC Method is not commercially available.  Three 
commercially available vitamin mixtures are pertinent to the PER study: (1) the AOAC vitamin 
mixture, which differs from the originally described AOAC Method vitamin mixture only in 
providing vitamins A and D together as a powder; (2) vitamin mixture AIN-76A; and (3) vitamin 
mixture AIN-93G (Appendix 4).  These vitamin mixtures are specified for use at 1% of diets.  
The three commercially available mixtures specify the forms of many of the vitamins, which was 
not done with the original AOAC Method formulation (see Appendix 1). 
The AOAC Method does not provide a formula to describe how to make adjustments in vitamin 
composition.  This may be because the use of the PER method with infant formulas or other 
foods that contained significant quantities of vitamins may not have been anticipated at the time 
the method was developed.  Furthermore, because there is not a single surrogate measure for all 
vitamins as there is for all minerals (i.e., ash), we are not aware of formulas that would 
encompass adjustments for all potential vitamin compositions of various formulas that might be 
tested.  Adjustments should be made on a formula-by-formula and vitamin-by-vitamin basis. 
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We recognize that the issue of comparability of vitamin compositions between the casein 
reference and infant formula test diets may be more complicated than that with mineral 
compositions, but it is still amenable to adjustment.  Our current thinking is that use of different 
amounts of a defined vitamin mixture in the reference and test diets may be one step toward 
ensuring matching levels of vitamins in both diets. 
Because most infant formulas provide significant quantities of vitamins as well as minerals, 
careful calculations should be made before diet formulation begins to determine the contributions 
of vitamins anticipated from the test formula and from the vitamin mixture(s).  In some cases, the 
infant formula itself may provide most or perhaps all vitamins, as per the NRCs Nutrient 
Requirements of Laboratory Animals (Reference 11).  Adjustments in amounts of vitamin 
mixture added to the infant formula-based test diet or preparation and use of a specific vitamin 
mixture to adjust for such a finding may be needed. 
Specific recommendations for several vitamins follow: 
  Vitamin E: Comments with respect to vitamin E as it relates to fat composition in 
the diets are found in Section IV.B.1.c., above. 
  p-aminobenzoic acid (p-ABA): The vitamin p-ABA is included in the AOAC 
vitamin mixture, but not in the AIN-76A or AIN-93G vitamin mixtures (Appendix 
4).  p-ABA, a vitamin that is a precursor of folic acid, is not needed by the rat and 
can be omitted from the vitamin mixture.  It may have been added to the original 
AOAC Method vitamin mixture to facilitate the synthesis of folate by intestinal 
bacteria. 
  Inositol: Inositol is included in the AOAC vitamin mixture, but not in the AIN-76A 
or AIN-93G vitamin mixtures (Appendix 4).  Inositol is a type of sugar that was 
once considered to be part of the B vitamin family and known as vitamin B8.  It can 
be synthesized in the animal body from glucose and is no longer considered to be a 
vitamin. Inositol is not required by rats under conventional conditions, but a 
requirement has been reported in lactating rats fed antibacterial drugs (Reference 
11).  If inositol is added to an infant formula, we recommend that it be included in 
vitamin mixtures used in PER studies of such formula to facilitate matching of 
inositol levels in the test diet. 
  Vitamin A: The estimates of vitamin requirements for rats have changed over the 
years.  This is most obvious in the concentrations of vitamin A included in vitamin 
mixtures and diets.  The AOAC Method vitamin mixture as listed in the AOAC 
Method and the AOAC vitamin mixture each provide 20,000 IU vitamin A per 
kilogram diet versus the current NRC (1995) (Reference 11) specification of 2,300 
IU per kilogram diet.  Infant formula typically has a high concentration of vitamin 
A.  Because of the importance of matching the reference and test diets, for infant 
formula, the concentration of vitamin A in the reference diet should be matched to 
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that of the test diet even if such matching results in reference diets of high vitamin A 
content.16 
  Vitamin K: Menadione (vitamin K3) is specified as the form of vitamin K in the 
AOAC Method vitamin mixture, and its use was continued in the AIN-76A vitamin 
mixture.  More recent vitamin formulations (e.g., AIN-93G vitamin mixture) 
include vitamin K as phylloquinone (vitamin K1).  Menadione is approximately 
one-tenth as active as phylloquinone.  We recommend that phylloquinone, rather 
than menadione, be used as the source of vitamin K.  The amounts and forms of 
vitamin K included in various vitamin mixtures are listed in Appendix 4. 
  Choline: Choline is an essential nutrient and serves as a methyl-donor in many 
physiological reactions.  While choline is not a vitamin, it is frequently listed with 
the vitamin component of diets (References 11, 14).  Choline (form unspecified) is 
included as a part of the AOAC Method vitamin mixture listed in the AOAC 
Method (Appendix 1).  Its dihydrogen citrate salt is used in the commercially 
available AOAC vitamin mixture (Appendix 4).  Choline is not included in the AIN-
76A and AIN-93G vitamin mixtures (Appendix 4).  Rather, the AIN-76A and AIN-
93G diet formulations include choline bitartrate as separate additions to the final 
diets.  The consequence of this change is that if the AIN-76A or AIN-93G vitamin 
mixtures are used to replace the AOAC Method vitamin mixture, and the need for 
the separate addition of choline is overlooked, the resulting finished casein reference 
diet will be deficient in choline.  The situation with respect to choline in the PER 
study test diet will need to be determined by evaluation of the choline content of the 
infant formula and its rate of addition in the test diet.  The levels of choline in the 
reference diet and in the infant formula test diet should be matched.  FDAs current 
thinking is that choline may need to be included as a separate addition to both diets. 
  Vitamin C: Vitamin C is not required by rats. It is included in infant formulas and 
will be present in PER study test diets.  The inclusion of vitamin C in PER study test 
diets is discussed in Section IV.B.1.i., below. 
The development of estimates of the compositions of the reference and test diets before 
beginning diet preparation, described in section IV.B.1.j. and Appendix 6, is applicable to 
matching vitamin compositions as well as mineral compositions. 
g.  Fiber 
The PER study diets described in the AOAC Method (Section III, above) include 1% fiber as 
cellulose.  For the test diet, this ingredient is calculated as 1% cellulose minus the fiber content 
 
16 If the AOAC Method is used for an optional, additional control group (i.e., a standard reference control group), it 
is possible to reduce the vitamin A in the vitamin mixture to a concentration closer to the current estimated 
requirement (i.e., 2,300 IU per kilogram diet). 
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of the test sample and is measured as crude fiber in the proximate analysis.  A review of 
Certificates of Analysis submitted with recent PER studies showed that AOAC Official Method 
962.09, a method for crude fiber in animal feed and pet food, is often used for determination of 
crude fiber. 
Rats do not require fiber in their diets.  The NRCs Nutrient Requirements of Laboratory 
Animals does not provide a value or a recommendation for fiber in rat diets and notes only that 
inclusion of fiber may be potentially beneficial (Reference 11).  However, although infant 
formulas do not contain cellulose, but do contain other fiber-like carbohydrates, we have 
considered whether to add cellulose to PER study diets conducted with infant formulas (i.e., to 
both the reference and test diets).17 
The NRCs Nutrient Requirements of Laboratory Animals (Reference 11) states: 
Some carbohydrates that cannot be properly called fiber also elicit some responses similar to 
those observed with true fibers.  Lactulose (disaccharide), raffinose (trisaccharide), and fructo-
oligosaccharides are not absorbed in the small intestine but are rapidly fermented in the 
hindgut. 
Infant formulas do not contain cellulose or other sources of crude fiber.  They do, however, 
frequently contain non-digestible carbohydrates such as fructo-oligosaccharides (FOS) or 
galacto-oligosaccharides (GOS).  Our current thinking is that it is not necessary to add cellulose 
to the infant formula-based test diet or to its matched casein reference diet when the infant 
formula includes non-digestible carbohydrates such as FOS and/or GOS.  When FOS or GOS are 
present in the infant formula, they should be included in the matched casein reference diet and 
analyzed by appropriate specific methods. 
Due to our uncertainty as to the outcome of PER studies conducted in completely fiber-free 
diets,18 we recommend that 1% cellulose be added to the test and matched casein reference diets 
 
17 In Section IV.B.1.f. and j., above, and in Appendix 6, we suggest additions to the infant formula-based test diet 
that are limited to those necessary to ensure that the test diet will meet the nutritional requirements of the rat (with 
the exception of protein).  In practice, the infant formula may provide the concentrations of certain minerals and 
vitamins in the test diet, which exceed the NRCs Nutrient Requirements of Laboratory Animals (Reference 11) and 
are much higher than those of casein reference diet in the AOAC Method.  In such situations, the reference diet 
should then be formulated to be comparable in all of its ingredients to the final test diet.  In general (see Section 
IV.B.1.j, below), we do not recommend the addition to the infant formula-based test diet of ingredients that are not 
initially present in the infant formula or those for which there is not a recommendation by the NRCs Nutrient 
Requirements of Laboratory Animals (Reference 11).  However, the question of whether fiber should be added to 
the test diet and matched casein reference diet under certain conditions raises this as a separate issue. 
18 While we have reviewed successful PER studies (i.e., those with PER values for the casein control group of 2.20  
0.14 g body weight gain per g protein consumed (see Reference 7)) in which no cellulose was added to the test and 
matched casein reference diet, the infant formulas that were the subjects of these submissions all contained either 
FOS or GOS.  Thus, both the test and matched casein reference group diets contained non-digestible carbohydrates.  
To date, we have not reviewed a PER study submission in which no source of non-digestible carbohydrate was 
included in the infant formula or no cellulose was added to the test and matched casein reference group diet (e.g., a 
PER conducted with fiber-free test and matched casein control diets). 
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when the infant formula does not contain a source of non-digestible carbohydrate.  We note that 
when Mitchell et al. (1985) (Reference 7) and Harris et al. (1988) (Reference 8) performed PER 
analysis on a variety of soy- and casein-based infant formulas, they added 1% cellulose to all test 
diets.  The infant formulas themselves did not contain complex carbohydrates. 
Finally, if a manufacturer and laboratories chooses to add an additional experimental group 
following the original AOAC Method (i.e., 10% protein, 8% fat; often identified as a standard 
casein control group), then 1% cellulose should be added to the diet for this group (See  
Appendix 1). 
h.   Sulfur Amino Acids (SAA) (methionine, cystine) 
We recommend that the PER casein reference control diets contain 0.964 g/kg inorganic sulfur 
as specified in the AOAC Method.  If there is concern about a potential failure of the casein 
reference group because of low concentrations of cystine in casein, manufacturers and 
laboratories may want to consider matching the sum of methionine plus cystine (i.e., methionine 
+ cystine) concentrations in the casein reference control and test diets.  If time and resources 
permit, manufacturers and laboratories may want to prepare the (methionine + cystine)-matched 
diet and include it as a second casein reference control group in their PER study. 
The following section provides background information to support these recommendations on 
the SAA methionine and cystine, then describes our current thinking with respect to an option  
for matching the concentrations of (methionine + cystine) in the control and test diets.  
Additional information regarding the concentrations of sulfur/sulfate salts in PER study diets is 
found in Section IV.B.1.e. 
Methionine and cystine  limiting amino acids in casein:  When an essential amino acid such 
as methionine is not provided in adequate quantities in a diet (e.g., in a protein-deficient diet), 
protein synthesis and growth are limited to the rate at which the essential amino acid is available. 
In such circumstances, the essential amino acid is referred to as the limiting amino acid.  In 
simpler terms, the first limiting amino acid is the essential amino acid that first becomes 
deficient in a diet.  The SAA methionine and cystine are first limiting in diets containing 8-10% 
protein from casein (Reference 20).  In addition, in casein, the concentration of cystine relative to 
that of methionine is quite low.  The concentration of cystine in casein (i.e., 0.42 g/16 g N) is 
only 14.3% of the concentration of methionine (i.e., 2.94 g/16 g N; Cys/Met x 100 = 14.3%) 
(Reference 21).  In comparison, the concentration of cystine in skim milk (i.e., 0.85 g/16 g N) is 
much higher relative to that of methionine (i.e., 2.32 g/16 g N; Cys/Met x 100 = 36.6%) 
(Reference 21). 
The rat requirement for SAA is expressed as the sum of methionine plus cystine (i.e., methionine 
+ cystine).  This value is 9.8 g/kg (0.98 g/100 g diet; 0.98%) for rats fed diets containing 15% 
protein (Reference 11).  While the corresponding value for rats fed 10% casein protein diets has 
not been determined, Peace et al. found that optimal feed-to-weight-gain ratio, relative net 
protein ratio, and plasma amino acid parameters were obtained when weanling rats were fed 8% 
casein plus amino acid diets containing 0.44% (0.44 g/100 g diet; 4.4 g/kg diet) total SAA with 
cystine replacing 33 to 60% by weight of dietary methionine (Reference 20).  The inclusion of 
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cystine at the expense of methionine in 8% protein diets improved overall rat performance and 
utilization of dietary methionine (Reference 20).  Provision of cystine may decrease the 
requirement for enzymatic conversion of methionine to cystine and its derivatives, permitting 
more efficient utilization of methionine for protein synthesis (Reference 20).  The ability of 
cystine to substitute for a portion of the methionine requirement is important because cystine is 
present in limited quantities in 10% casein diets.  Thus, the contribution that cystine can make to 
overall methionine metabolism is reduced in rats fed 10% casein protein diets. 
SAA and sulfur/sulfate content:  The AOAC Method does not include the addition of cystine.  
To better understand why, we evaluated the composition of the mineral mixtures used in the diet.  
The AOAC Method mineral mixture includes several sulfate salts, and the diet, when prepared 
with 5% of the mineral mixture, provides 0.964 g sulfur/kg.  Use of the Bernhart and Tomarelli 
mineral mixture gives PER results comparable to those obtained with use of the mineral mixture 
described in the AOAC Method. The Bernhart and Tomarelli mineral mixture provides 0.501 g 
sulfur/kg when used at 4% (Appendix 3).  The original AOAC Method mineral mixture and the 
Bernhart and Tomarelli mineral mixture both provide considerably more sulfur than provided by 
either the AIN-76A or AIN-93G mineral mixtures (e.g., 0.337 and 0.301 g sulfur/kg, 
respectively, when used at 3.5%) (Appendix 3). 
The diets of PER study control groups for which we have data (see below Section IV.B.2) used 
either the AOAC Method mineral mixture or the Bernhart and Tomarelli mineral mixture.  Both 
mixtures provided sulfur at concentrations considerably higher than the value of 0.3 g/kg diet 
identified in the current NRC (1995) Nutrient Requirements of Laboratory Animals (Reference 
11) as potentially beneficial in diets of 15% protein.  The increased inorganic sulfur from the 
AOAC Method mineral mixture or the Bernhart and Tomarelli mineral mixture may have 
contributed significantly to sulfur metabolism and compensated for the low SAA content of the 
10% casein diets.  Under such circumstances, a separate addition of cystine was not needed in 
the original AOAC Method casein reference control diet. 
While we do not have data that would assist in making a precise evaluation regarding addition of 
inorganic sulfate in the PER study casein reference control diets in studies with infant formulas, 
our current thinking is that inorganic sulfur should be included in diets at levels like those found 
in PER study control diets described above (i.e., between 0.50 and 0.96 g/kg diet).  Our current 
thinking is that a concentration at the high end of the 0.50 and 0.96 g/kg range should be used as 
a target concentration because it will provide a margin of safety.  Therefore, we recommend that 
the concentration of inorganic sulfur in the PER study casein reference control diets when used 
with infant formulas be adjusted to provide the inorganic sulfate content set forth in the AOAC 
Method (i.e., 0.964 g/kg diet). 
Possible addition of cystine:  The information above suggests that in PER study diets with 
significant inorganic sulfur content (e.g., 0.50-0.96 g/kg, or 0.05-0.096%), the addition of cystine 
may not be needed.  However, some losses of nutrients (e.g., due to diarrhea) may occur based 
on the composition of the diet (e.g., high lactose), and under such conditions, a low cystine 
concentration in the casein reference control diet (e.g., perhaps 20-30% of the value in the test 
diet) might cause a growth failure in the group.  This suggests that the addition of cystine to the 
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casein control diet sufficient to match the amount of (methionine + cystine) in the test diet may 
be considered. 
Matching the concentrations of (methionine + cystine) in PER study test and casein 
reference diets and inclusion of the group as a second reference control group in PER 
studies:  Manufacturers and laboratories may want to consider matching the concentrations of 
(methionine + cystine) in the PER study test and reference casein diets if there is concern about a 
potential failure of the casein reference group because of low concentrations of cystine in casein.  
The following paragraphs describe a process by which this matching may be accomplished. 
The infant formula and the casein for the control diet can be analyzed for their methionine and 
cystine content prior to diet preparation.  The concentrations of (methionine + cystine) can be 
calculated from the results of these analyses.  The calculation can be corrected for the rate of use 
of the infant formula in the PER study test diet.  A similar calculation can be made from the 
amino acid composition information for the casein that will be used in the control diet.  If the 
analyzed concentration of cystine in the control diet is < 80% of the value for cystine in the test 
diet (calculated from the analyzed value in the infant formula and corrected for its rate of use), 
then cystine can be added to the control diet so that the concentrations of (methionine + cystine) 
are similar in both diets.  In practice, this will generally involve only calculation of the amount of 
cystine to be added to the control diet.  The final concentration of (methionine +cystine) in the 
control diet will be within   20% of the value for (methionine + cystine) in the test diet (see 
Appendix 6). 
If time and resources permit, manufacturers and laboratories may want to prepare the 
(methionine + cystine)-matched diet described above and include it as a second casein reference 
control group in their PER study.  The first control group would meet FDA recommendations 
regarding modifications with respect to matching fat types, lactose, vitamins, minerals, and other 
nutrients, and would contain the amount of inorganic sulfur specified in the AOAC Method.  
This primary casein reference control group is usually referred to as a Modified Casein Control 
Group.  The second control group would have the same composition as the first and, 
additionally, would be matched for concentrations of (methionine + cystine) in the control and 
test diets.  Comparison of PER results with these two groups will assist in determining the 
relative importance of the need for increased inorganic sulfur versus increased SAA in the PER 
study casein reference diets. 
Lack of justification for addition of methionine:  We are unaware of a justification for adding 
methionine alone to raise the (methionine + cystine) concentration in the control diet above the 
level of (methionine + cystine) in the test diet because there is insufficient information from 
which to derive an estimate of the concentrations of total SAA or of methionine alone that are 
needed for 10% casein diets.  However, the steps outlined above will ensure that the test and 
control diets are matched within  20% in their contents of (methionine + cystine).  The purpose 
of the addition of cystine is to increase the cystine content of the casein control diet so that the 
(methionine + cystine) contents of both diets are similar.  We are uncertain as to whether the 
addition of methionine alone will suffice to remedy the limited concentration of cystine in the 
casein control diet.  In general, concentrations of methionine among proteins are more similar 
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than are concentrations of cystine.  In some instances, the concentration of methionine in the 
casein control diet may be higher than that in the infant formula-based test diet.  In such a case, if 
addition of methionine only were under consideration, then addition of methionine would appear 
unnecessary.  However, the concentrations of cystine in the test and control diets (and the values 
for (methionine + cystine)) would remain unmatched. 
We note that DL-methionine was the recommended supplement to the AIN-76A diet.  During the 
formulation of the AIN-93 diets, the AIN noted that compared with other milk proteins, L-
cysteine or L-cystine and not L-methionine were the amino acids found in small amounts in 
casein. For this reason, the AIN recommended L-cystine instead of L-methionine as the 
supplement to the AIN-93 diets (Reference 22). 
In cases in which the concentration of (methionine + cystine) in the test diet is less than the 
corresponding sum in the control diet, and the concentration of cystine in the control diet is < 
80% of the concentration of cystine in the test diet, then sufficient cystine can be added to the 
control diet to match its concentration in the test diet. 
Our current thinking is that the approaches described above will reduce the risk of a failure of the 
PER study casein reference control group. 
i.  Other Constituents (including vitamin C) 
Other constituents:  Infant formulas may contain other components (e.g., nucleotides, taurine, 
oligosaccharides) that would not normally be included in rat diets.  Our current thinking is that 
the composition of the PER study reference diet should be matched as closely as possible to that 
of the infant formula-based test diet.  Concentrations of constituents such as inositol, carnitine, 
taurine, nucleotides, docosahexaenoic acid (DHA), arachidonic acid (ARA), GOS, or FOS, or 
others in the infant-formula-based test diets should be evaluated, and such constituents should be 
included in the PER study reference diet at the same concentrations found in the test diet.  
Results of the analyses by appropriate and specific methods of components that are added to the 
PER study casein control diet to match their concentration in the infant formula test diet should 
be included in the final report of the PER study. 
Vitamin C:  Infant formulas must contain vitamin C, and it will be present in the PER study test 
diets.19  Vitamin C is not needed by the rat.  Vitamin C should be included in the PER study 
reference diet at a concentration equivalent to its concentration in the PER study test diet. 
j.  Developing Estimates of Compositions of PER Study Reference and Test Diets 
(minerals and vitamins) 
In Appendix 6, we recommend an approach that can be used to develop the compositions of PER 
study test and reference diets from the composition of the infant formula that is the subject of the 
 
19 See 21 CFR 107.100 
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submission.  The first step using this approach is to list all components of the infant formula in 
units/100 g or units/kg.  The rate of addition of the formula to the test diet will provide the 
starting point for formulation of mineral and vitamin premixes.  The sum of nutrients provided 
by the infant formula at its rate of addition and contributions from mineral and vitamin mixtures 
will provide information to formulate the reference diet.  We recommend matching the mineral 
and vitamin compositions of the PER study reference diet within  20% of the mineral and 
vitamin compositions of the PER study test diet. 
In Appendix 6, we recommend a format that may be useful for developing estimates of the 
mineral and vitamin compositions of the test and reference diets.  In addition, we provide worked 
examples, which includes values for parameters, for several minerals and vitamins and show 
how to view the information to provide a comparison of nutrients in both the test and reference 
diets.  Review of the NRCs Nutrient Requirements of Laboratory Animals for rats at various 
steps of diet development (e.g., during formulation of premixes, during diet preparation) will be 
helpful in assessing the adequacy of the test and reference diets (Reference 11). 
k.  Chemical Analyses (demonstrating the appropriateness of modifications) 
The AOAC Method stipulates proximate analyses (e.g., for nitrogen, fat, ash, moisture, and 
crude fiber) be performed to match major components of the PER study test and reference diets.  
Calculations of anticipated diet compositions should be performed to determine what additional 
analyses might be appropriate.  Following such comparisons, FDA recommends that 
manufacturers and laboratories analyze several vitamins and minerals to ensure that the diet 
compositions are as anticipated.  Analysis of samples from different locations in the mixing bowl 
or other mixing apparatus (e.g., top, middle, and bottom of a mixing container) is useful for 
assessing homogeneity of diet preparation.  Consideration should also be given to analysis of 
vitamins that may be present in different forms in the test and control diets (e.g., vitamin K). 
A demonstration that appropriate modifications have been made includes documentation that 
proximate analyses have been performed and that appropriate modifications were addressed in 
the diets.  In addition, when full compositions of the PER study test and reference diets are 
provided to FDA (e.g., in a protocol or in the PER study final report), it is possible for us to 
confirm by calculation that appropriate modifications were made.  Our current thinking is that 
the inclusion of a full specification (e.g., a Certificate of Analysis (CoA)) for the lot/production 
run of infant formula used in the PER study with nutrients expressed as units/100 g or units/kg 
and minerals expressed as their elemental concentrations (i.e., calcium, not calcium carbonate) is 
essential for use in verifying the accuracy of the composition of the test diet.  The addition rate 
of the formula to the PER study test diet should be stated in the diet preparation records.  
Complete diet preparation records should be summitted with the protocol and final report.  A 
manufacturer may wish to include examples of calculations used in preparing the PER study 
diets, and we encourage the inclusion of this information.  Inclusion of records of all chemical 
analyses and their methods and results also are also helpful in determining the appropriateness of 
the compositions of the PER study test and reference diets. 
 
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2.  PER Values for Casein Reference Control Diets 
The following paragraphs describe our current thinking on attainable values for casein reference 
control groups when the AOAC Method is performed as originally described, when the AOAC 
Method is performed with appropriate modifications with lactose-free infant formulas, and when 
the AOAC Method is performed with appropriate modifications with high-lactose infant 
formulas. 
Casein reference control data  Original conditions of the AOAC Method: The available 
data show that when the AOAC Method is performed as described (including, among other 
components, 10% protein, 0% lactose, 8% fat, and 5% AOAC-specified mineral mixture), 
additional cystine is not needed, and sustained growth can be achieved in the control group over 
the 28-day bioassay period.  FDA considers that PER values for the casein control group 
between 2.62 and 3.09 g body weight gain per g protein consumed over the 28-days are 
attainable under the original conditions of the AOAC Method, which includes use of a mineral 
mixture providing 0.50-0.96 g sulfur/kg diet (see Footnote 6 for data and Appendix 3 for 
compositional information). 
Casein reference control data  AOAC Method modified for infant formulas  Lactose-
free formulas:  When the AOAC Method is appropriately modified for use with infant formulas 
and lactose-free infant formulas are studied, with PER diets containing, among other 
components, 10% protein, 0% lactose, and 21-28% fat, PER values for the casein control group 
between 2.62 and 3.09 g body weight gain per g protein consumed over the 28-days are 
attainable. 
Casein reference control data  AOAC Method modified for infant formulas  High-
lactose formulas:  We have also identified attainable PER values when the AOAC Method is 
appropriately modified for use with infant formulas and high-lactose infant formulas are studied.  
The limited data available were obtained from studies in which the AOAC Method was 
performed with infant formulas with diets including, among other components, 10% protein, 
43% lactose, 24% fat, and 4% of the Bernhart and Tomarelli mineral mixture (Appendix 3; 
Reference 7) for a 28-day bioassay period.  Cystine was not added to the control group diet in 
these studies.  FDA considers that PER values for the casein control group of (mean  SD; n=2) 
2.2  0.14 g body weight gain per g protein consumed over the 28-day bioassay period (range 
2.06-2.36) are attainable under dietary conditions of high lactose and high fat when mineral 
mixtures are used that provide 0.50-0.96 g sulfur/kg diet (Appendix 3). 
Summary:  FDA considers the following ranges of PER values (g weight gain/g protein 
consumed for 28 days) for casein reference control groups to be attainable: 
  Under original conditions of the AOAC Method:  2.62  3.09 
  Under modified conditions for infant formulas:  Lactose-free formulas:  2.62 - 3.09 
  Under modified conditions for infant formulas:  High-lactose formulas:  2.06  2.34 
 
 
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3.  Experimental Animals 
a.  Age, Weight, Number/Group, Acclimation Period, Housing, Acclimation Diet 
Age and Weight.  The AOAC Method specifies that experimental animals be weanlings  21 
days of age but  28 days of age.  The range of individual rat weights among animals used 
should be  10 grams.  FDA recommends that these ranges be maintained to limit the variability 
associated with the bioassay. 
Number/Group.  Groups of  10 rats are needed for a PER study.  A reference group that will 
receive the casein reference diet is needed for each test formula group.  If the test formulas are 
sufficiently similar, one reference casein group can be used for a concurrent assay of more than 
one test material.  As stated in the AOAC Method, when assembling all groups is complete, the 
total number of rats in each group should be the same, and the average weight of rats in any one 
group at the beginning of the assay period should not exceed by > 5 grams the average weight of 
rats in any other group (see also Appendix 1). 
A randomization method is not specified in the AOAC Method, but several procedures are 
available (e.g., stratification by weight followed by randomization; or use of randomization 
statistical functions in commercially available software). 
Acclimation Period.  The AOAC Method specifies acclimation periods of  3 days, but < 7 
days.  Most experimental animals are transported from breeding facilities to the sites where the 
studies will be carried out.  FDAs current thinking is that acclimation periods of 1 to 2 days are 
usually needed to acclimate the young animals to their new surroundings.  Several additional 
days, running concurrently, may be needed to acclimate the young animals from nursing to 
individual feeders and from milk to solid diets. 
Housing.  Housing animals individually during acclimation, while not specified in the AOAC 
Method, will facilitate the transfer to individual housing and individual feeding that is required 
during the 28-day PER study experimental period (Reference 1).  Housing in wire-bottom cages 
throughout will reduce coprophagy (i.e., eating of feces).20 
Acclimation Diets.  Acclimation diets are not specified in the AOAC Method, and specific 
literature on acclimation diets is limited.  We recommend feeding diets of 10% casein (e.g., such 
as a PER study reference diet) to both the test and control groups during the acclimation period 
(Reference 23).  The alternative of feeding diets with high protein levels (e.g., chow-type diets, 
generally  16% protein) will likely bring in high levels of minerals and vitamins, and this 
exposure may make it more difficult for the rats to adjust to their PER study diets.  Rats should 
 
20 Feces may contain several vitamins synthesized by intestinal bacteria.  Consumption of feces is a practice which 
can provide such vitamins to the animal. 
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be weighed at the beginning and the end of the acclimation period, and the records should be 
maintained with the study records. 
The use of infant formulas in PER studies warrants further consideration regarding a suitable 
acclimation diet.  Our current thinking and recommendations are described in the following 
paragraphs. 
Certain carbohydrates may have detrimental effects on growth and PER values when they are 
present in high concentrations and may result in test samples appearing to be lower in protein 
value than their true value.  Lactose is present in milk-based formulas and is added as a separate 
ingredient to other formulas.  As discussed in Section IV.B.1.a, FDA recognizes that appropriate 
modifications to the AOAC Method are needed to address the adverse effect of lactose in rats, 
and the need to have PER reference and test diets matched in lactose content (that is, the 
reference diet must contain a level of lactose comparable to that of the test diet). 
While the need for matching the levels of lactose in PER reference and test diets is well 
recognized, there is little information regarding the need (or not) for lactose in diets fed during 
the acclimation period (References 7, 24).  Mitchell and Jenkins, in their studies with infant 
formulas, did not provide lactose-containing diets during their short (2-day) acclimation periods 
(Reference 7).  While Burnette and Rusoff recommended that the feeding of acclimation diets 
containing 20% lactose should precede the feeding of test diets containing more than 20% 
lactose, they did not provide data showing the results of such acclimation on results of a 
subsequent PER study (Reference 24). 
DeAngelis et al. conducted studies in which weanling rats (21 days old) were fed diets 
containing 10% protein and varying levels of lactose (0, 1, 2, 5, 10 , 20 and 50%) for 15 days  
(Reference 25).  They observed that PER values (at 15 days) at 10% and 20% lactose were not 
different from those obtained under control conditions.  They noted that growth of weanling rats 
was normal when lactose in diets was present at levels < 20%.  When dietary lactose was 50%, 
PER values (at 15 days) decreased to 0.92  0.45 (mean, standard deviation) (g weight 
gain/protein ingested) from PER values of 3.51  0.15 for 0% lactose and 3.04  0.19 for 20% 
lactose.  No acclimation period was reported in this 15-day study.  While these data suggest that 
rats tolerate lactose at levels  20% , they do not address the need for lactose in acclimation 
diets. 
The high intestinal lactase activity in neonatal rats declines rapidly around the time of weaning 
(Reference 26).  Van de Heijning et al. reported that in neonatal Wistar rats, intestinal lactase 
activity decreased promptly upon weaning (post-natal day 21) and, when the weaned animals 
were placed on diets containing 30% lactose, remained at a low residual level (about 25%) into 
adulthood (Reference 26).  Their study did not corroborate the theory that keeping the substrate 
available can lead to maintenance of newborn lactase levels.  Their work may also suggest that 
adaptation to high lactose diets does not occur. 
On the basis of the limited literature available, we recommend that one of the two options be 
used for acclimation diets: 
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(1) Use an acclimation diet containing 20% lactose for an infant formula containing  
20% lactose.  The  20% lactose diet can be used to acclimate both the test and 
reference groups; or 
(2) Use an acclimation diet that matches the lactose level in the test diet.  As a 
practical issue, in this case, the casein reference diet can be used as the acclimation 
diet for both the test and reference groups. 
We do not have enough information to recommend whether the acclimation diet should contain 
20% lactose or a higher level that matches that of the test group diet.  The Van de Heijning et al. 
data suggest that there is little adaptation to high lactose diets, and for this reason, we 
recommend that acclimation periods be as short as possible (perhaps only as long as is required 
for the weaning rats to adjust to individual housing and a non-milk diet) (Reference 26).  We 
recommend that manufacturers review their PER studies and, based on their own experience, 
decide which of these two options is more appropriate for them.  We currently consider either 
approach sufficient to facilitate the adaptation of the rats to their high-lactose PER study diets. 
b.  Assay Period, Type, and Frequency of Measurements 
The AOAC Method specifies an assay period of 28 days with food and water available ad 
libitum throughout.  Environmental conditions for test and reference casein groups should be 
monitored and maintained as uniformly as possible.  Body weights of each rat should be 
recorded on the first day of the assay period.  Body weights and food intakes of each rat should 
be measured at regular intervals, ideally daily but at intervals not greater than 7 days, and on the 
28th day after the beginning of the assay period.  Measurement of body weights and food intakes 
at more frequent intervals (e.g., twice per week) are useful with new formulas so that decreases 
in weight gain or food intake may be identified promptly. We recommend further that during 
each collection of live animal data, the new records be compared with data collected previously 
to confirm that patterns of food intake and body weight are as expected. The potential utility of 
measuring water consumption as well as food consumption should be considered. 
c.  Monitoring Attrition and Adverse Effects of Diets 
Dietary components such as lactose, unusual oils, or proteins of low quality may cause changes 
in the growth of rats during the acclimation phase and experimental phase of a PER study.  For 
these reasons, FDA recommends that attrition and adverse effects (e.g., oily coats, staining on 
hair, loose stools, diarrhea, cataracts) of the PER study diets on the reference casein and test 
group rats be monitored and recorded at regular intervals, ideally daily, but not greater than 
every 7 days.  All such results should be included in the final report of the PER study.  Such 
adverse effects may serve as an early warning of potential problems with components of the 
infant formula. 
 
 
 
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4.  Tabulation and Calculation of Results (e.g., statistical analyses; reference data; 
record-keeping) 
The AOAC Method specifies that average 28-day weight gains, protein intake, PER (g body 
weight gain/g protein consumed during the 28-day test period), and ratio x 100 of sample PER to 
reference casein PER should be tabulated for each group.  In addition, FDA recommends that the 
28-day weight gain, the food intake and protein (N x 6.25) intake, and the PER (g weight gain/g 
protein intake) be recorded and calculated for each rat. 
The AOAC Method does not specify statistical analyses to be conducted.  Thus, statistical 
analysis of PER data can be performed at the discretion of the laboratory performing the PER 
study or the manufacturer that reviews the results.  Commonly used statistical analyses include 
the calculation of mean and standard deviations for all continuous data, including overall and 
weekly body weights and body weight gains, overall and weekly food and protein consumptions, 
and the PER values.  Analysis of variance is frequently used to compare the PER of the test 
group to that of the casein reference group.  A variety of statistical programs are available for 
this purpose.  FDA encourages performing statistical analysis. 
Unlike growth monitoring studies for infants, there are no growth reference data for the rat PER 
studies.  However, FDA recommends a review of the PER literature (e.g., PER values, diet 
compositions) found in Section VI (References) to determine whether unexpected results have 
been obtained. 
Recordkeeping is essential to understanding the details of the conduct of a PER study.  This is 
particularly important in the areas of diet preparation and in details of parameters presented in 
the AOAC Method as a range.  For example, the ages and weights of rats on arrival should be 
recorded as individual values (rather than as broad ranges) and the individual data should be 
retained as part of the study records. 
V.  Miscellaneous 
A. Protocols and Reports 
The AOAC Method does not mention development of a protocol or final report.  We recommend 
that both be prepared.  Manufacturers with in-house facilities and contract laboratories with 
experience performing PER studies may have a protocol form available that can be used to 
develop a draft for the conduct of the desired PER study.  Such a draft protocol should be 
developed to ensure that the specifications of the AOAC Method and FDAs appropriate 
modifications are met.  The benefit of chemical analyses in addition to proximate analyses to 
confirm critical aspects of PER study diet preparations may become apparent during protocol 
development. 
While not required, we recommend that the protocol be reviewed by us before initiation of the 
PER study.  Manufacturers that are interested in sending a protocol for review may contact the 
Infant Formula and Medical Foods Staff (IFMFS) at (240) 402-1450.  After protocols are 
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provided to the IFMFS, reviewers may analyze aspects of the protocol, including the plans for 
diet preparation, plans for matching test and control diets, proposals for acclimation diets, 
proposals for data collection, and records to be maintained.  In the review, IFMFS may identify 
areas that the manufacturer or laboratory may want to reconsider such as lack of appropriate 
matching between control and test diets, proposed use of acclimation diets that do not seem to 
match either the test or control diets, or situations in which we cannot identify the origin of 
specific nutrients in the diets.  Instances may be identified in which specific pieces of 
information are missing, or where calculations appear to be inaccurate.  IFMFS will then provide 
feedback to the requestor. 
We recommend that complete records for all aspects of a PER study be maintained and that a 
final report be prepared.  The report should include the following: 
1.  The full specification for the infant formula under consideration, with nutrient 
composition expressed as units/100g or units/kg (i.e., quantitative formulation and 
nutrient content); 
2.  Information related to calculation of compositions of PER study acclimation, 
reference, and test diets; and 
3.  Information on the conduct of the live animal phase of the study, including selection 
criteria for rats upon arrival (e.g., by random numbers), selection criteria for rats when 
assigned to study groups (e.g., stratified randomization procedures), and descriptions 
of animal husbandry practices (e.g., type of caging, feeders and water bottles or 
watering system, animal room temperature and humidity). 
The usefulness of weekly measurements of water consumption should be considered.  Our 
current thinking is that laboratories performing PER studies should be accredited by the 
Association for Assessment and Accreditation of Laboratory Animal Care International 
(AAALAC) and should adhere to the Guide for the Care and Use of Laboratory Animals 
(Reference 27).  Documentation of the nature and frequency of live animal observations 
(including those made during the acclimation phase) should be included in the final report.  
Other important records that should be included in the final report include all individual live 
animal data (body weights and food consumption, including food consumption during the 
acclimation phase), individual protein consumption values for each animal, results of all 
chemical analyses related to diet compositions (proximate and other analyses), and calculations 
of individual and group PER values. 
The full specification for the infant formula (i.e., quantitative formulation and nutrient content) 
for the lot/production run of the infant formula that will be used in the PER study and anticipated 
compositions for the PER study control and test diets should be included in the PER study 
protocol as well as in the final report.  The availability of the complete specification (e.g., CoA) 
for the batch/lot of infant formula that will be used in the PER study is critical because numerous 
calculations are developed from it. 
 
 
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B. Reference Guidelines 
Nutrient compositions of rat diets vary according to experimental objectives, and many practical 
diets may include nutrients at levels that exceed specifications as a margin of safety.  However, 
the purpose of the PER study is to compare protein quality with as few confounding variables as 
possible and to provide a short-term diet that meets or exceeds all rat estimated nutrient 
requirements (Reference 11), rather than to provide an optimized diet. 
We recognize that there is limited information on the specifications for all nutrients in 10% 
protein diets.  As an example of how NRCs Nutrient Requirements of Laboratory Animals 
specifications may differ in diets of low protein content, Peace et al. reported that the sulfur-
amino acid requirements for rats fed 8% protein were 0.33% of the diet or 4.1% of dietary 
protein, rather than the value of 0.98% (DL-methionine + cystine) of the NRC diet (References 
11, 20).  We consider that most nutrient levels would be significantly lower for the 10% protein 
PER study diets than NRC values for 15% protein diets, but we are not able to determine the 
reductions that would be appropriate for each nutrient.  Thus, we are unable to provide guidance 
on how best to use the levels provided in NRCs Nutrient Requirements of Laboratory Animals 
in such circumstances (Reference 11).  These levels should be used with caution and should not 
be used to adjust the overall compositions of the PER study diets (Reference 11).  Rather, they 
should be used to identify and correct a PER study diet that may be deficient in a specific 
vitamin or mineral and to identify and correct a PER study diet that may be formulated to contain 
a potentially toxic level of a specific mineral or vitamin (see, e.g., section IV.B.1.c). 
   
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VI.  References 
The following references marked with an asterisk (*) are on display at the Dockets Management 
Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 
20852, 240-402-7500, and are available for viewing by interested persons between 9 a.m. and 4 
p.m., Monday through Friday; they also are available electronically at 
https://www.regulations.gov.  References without asterisks are not on public display at 
https://www.regulations.gov because they have copyright restriction.  Some may be available at 
the website address, if listed.  References without asterisks are available for viewing only at the 
Dockets Management Staff.  FDA has verified the website addresses, as of the date this 
document publishes in the Federal Register, but websites are subject to change over time. 
1.  Official Methods of Analysis of AOAC International, 18th edition, 1995, AOAC Official 
Method 960.48 Protein Efficiency Ratio, Rat Bioassay, Section 45.3.04.* 
2.  Hegarty, F.V.J.  1975. Some biological considerations in the nutritional evaluation of 
foods.  Food Technology. April 1975, pp. 52-64. 
3.  3.  Bos, C., Gaudichon, C. and Tome, D. 2000. Nutritional and physiological criteria in the 
assessment of milk protein quality for humans, Journal of the American College of 
Nutrition. 19 (2): 191S-205S. 
4.  Steinke, F.H.  1977. Protein efficiency ratio  Pitfalls and causes of variability: A review. 
Cereal Chemistry.  54: 949-957. 
5.  Hackler, L.R.  1977.  Methods of measuring protein quality: A review of bioassay 
procedures.  Cereal Chemistry. 54 (4):984-995. 
6.  Staub, R.W. 1978. Problems in evaluating the protein nutritive quality of complex foods. 
Food Technology. 32 (12): 57-61. 
7.  Mitchell, G.V. and Jenkins, M.Y. 1985. Assessment of protein quality methodology for 
infant formulas.  Journal of the Association of Official Analytical Chemists. 68 (4): 680-
683.   
8.  Harris, D.A. and Burns, R.A. 1988. Evaluation of infant formula protein quality: 
Comparison of in vitro with in vivo methods. Journal of the Association of Official 
Analytical Chemists. 71 (2):  353-357. 
9.  Derse, P.H. 1960. Evaluation of protein quality (Biological Method). Journal of the 
Association of Official Analytical Chemists. 43(1): 38-41. 
10.  Derse, P.H.  1965.  Vitamins and Other Nutrients: Evaluation of Protein Quality 
(Biological Method).  Journal of the Association of Official Analytical Chemists. 48(4): 
847-850. 
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11.  The National Academies of Sciences, Engineering, and Medicine, National Research 
Council (NRC), 1995, Nutrient Requirements of Laboratory Animals. Chapter 2, Fourth 
Revised Edition, 1995; http://www.nap.edu/catalog/4758.html 
12.   Chapman, D.G., Castillo, R., and Campbell, J.A. 1959. Evaluation of protein in foods. I. A 
method for the determination of protein efficiency ratios. Canadian Journal of 
Biochemistry and Physiology. 37(5): 679-686. 
13.  Harris, P.L. and Embree, N.D. 1963. Quantitative consideration of the effect of 
polyunsaturated fatty acid content of the diet upon the requirements for vitamin E. 
American Journal of Clinical Nutrition 13: 385-392. 
14.  The National Academies of Sciences, Engineering, and Medicine, National Research 
Council (NRC), 1978, Nutrient Requirements of Laboratory Animals. Third Revised 
Edition, 1978. 
15.   Hopkins, D.T. and Steinke, F.H. 1976. Effect of water of hydration on the measurement of 
the protein efficiency ratio of casein and soybean protein in rats. Journal of  Nutrition 106: 
1438-1446. 
16.  Steinke, F.H., Prescher, E.E., and Hopkins, D.T. 1980. Nutritional evaluation (PER) of 
isolated soybean protein and combinations of food proteins. Journal of Food Science 45: 
323-327. 
17.  Bernhart, F.W. and Tomarelli, R.M.  1966. A salt mixture  supplying the National Research 
Council estimates of the mineral requirements of the rat. Journal of Nutrition 89: 495-500. 
18.  Michels, F.G. and Smith, J.T.  1965. A comparison of the utilization of organic and 
inorganic sulfur by the rat. Journal of Nutrition 87: 217-220. 
19.  The National Academies of Sciences, Engineering, and Medicine, National Research 
Council (NRC), 1963. Warner, R.G. Nutrient Requirements of the Laboratory Rat. National 
Research Council Committee on Animal Nutrition, publication 990. National Academy of 
Sciences, Washington, DC. 
20.  Peace, R.W., Sarwar, G., Botting, H.G., and Chavez, E.R. 1985. Sulfur amino acid 
requirements of the growing rat fed eight percent dietary protein. Nutrition Research 6: 
295-307. 
21.  Sarwar, G., Peace, R.W. Botting, H.G. and Brule, D.  1989.  Relationship between amino 
acid scores and protein quality when based on rat growth.  Plant Foods for Human 
Nutrition  39: 33-44. 
22.  Reeves, P.G., Nielsen, F.H., and Fahey, G.C. Jr. 1993.  AIN-93 Purified diets for laboratory 
rodents: Final report of the American Institute of Nutrition Ad Hoc Writing Committee on 
the reformulation of the AIN-76A rodent Diet. Journal of Nutrition 123:1939-1951. 
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23.  Hackler, LR., Bodwell, C.E., Happich, M.L., Phillips, J.G., Derse, P.H., Elliott, J.G., 
Hartnagel, R.E., Hopkins, D.T., Kapiszka, E.L., Mitchell, G.V., Parsons, G.F., Prescher, 
E.E., Robaidek, E.S., and Womack, M.  1984. Protein Efficiency Ratio: AACC/ASTM 
Collaborative study. Journal of the Association of Official Analytical Chemists. 67 (1): 66-
77. 
24.  Burnette. M.A. and Rusoff, I.I. 1978. GMA test protocol for protein quality assays. Food 
Technology, December 1978, pp. 66-68. 
25.  DeAngelis, R.C., Guili, G.G., Rogano, R.N. and Terra, I.C.M.1983. Lactose load diet effect 
in rats. Arquivos de Gastroenterologia. S. Paolo, 20 (4) 166-169. 
26.  Van de Heijning, B.J.M., Kegler, D., Schipper, L., Voogd, E., Oosting, A. and van der 
Beck, E.M.  2015. Acute and chronic effects of dietary lactose in adult rats are not 
explained by residual intestinal lactase activity. Nutrients, 7: 5542-5555. 
27.  Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2001, National 
Academies Press, Washington, DC. 
   
34  Contains Nonbinding Recommendations 
Draft-Not for Implementation 
VII. Appendices
Appendix #1.  AOAC Official Method 960.48 
Appendix #2.  Adjustments required by the Association of Official Analytical Chemists AOAC 
Official Method 960.48. 
Appendix #3.  Contributions to diets (g/kg) of specific mineral mixes used at listed levels. 
Appendix #4.  Contributions to diets (units/kg) of specific vitamin mixes used at 1%. 
Appendix #5.  Compositions of the Association of Official Analytical Chemists (AOAC) 
Official Method 960.48 diet, diets AIN-76A and AIN-93G and National Research 
Council (NRC) Nutrient Requirements for Rats (1978 and 1995). 
Appendix #6.  Matching the mineral,  vitamin, and amino acid compositions of PER study test 
and reference diets. 
35  Appendix #1. AOAC Official Method 960.48 
45.3.04  Table 960.48.  Vit am  in mixture 
AOAC Off i cial Method 960.48 
Ing red ie nt  mg/100 g ra tion 
Prot ein Ef fi ciency  Ra tio 
Vit am  in A (dry, stab il ized)  2000 (IU) 
Rat Bioassay 
First Act ion 1960  Vit am  in D (dry, stab il ized)  200 (IU)
Fi nal Ac tion 1962  Vit am  in E (dry, stab il ized)   10 (IU) 
(Ap pli ca ble to ma te ri als con tain ing  >1.80% N.)  Menadione  0.5 
A. Re agents  Choline  200 
(a) ANRC ref er ence ca sein.Availa ble from New Zeal and  p-Amin ob enz oic  acid  10 
Milk Prod ucts (1269 N. McDowell, PO Box 80816, Petaluma, CA  Inositol  10 
94975-8016, USA).  Ni a cin  4 
(b) Salt mixt ure USP.Eit her USP salt mixt ure or salt mixt ure 
Ca-D-pantothenate  4 
hav ing es sen tially  same pro por tions of the el e ments. Pre pare USP 
Ri bo fla vin  0.8 
XIX (p. 612) salt mix ture (or cor re spondi ng USP XX item) as 
fol lows: Grind in mort ar por tion of 139.3 g NaCl with 0.79 g KI.  Thi a mineHCl  0.5 
Simi  larly grind to gether re main der of the NaCl with 389.0 g  PyridoxineHCl  0.5 
KH2PO4, 57.3 g MgSO4 an hy drous, 381.4 g CaCO3, 27.0 g  Fo lic acid  0.2
FeSO47H2O, 4.01 g MnSO4H2O, 0.548 g ZnSO47H2O, 0.477 g  Bi o tin  0.04
CuSO5HO, and 0.023 g CoCl 6HO, fi nally add ing the NaClKI 
4 2 2 2 Vit am  in B 0.003 
12 
mixt ure. Re duce en tire mix ture to fine powd er. 
Gluc ose to make  1000 
(c) Vi ta min mix ture.See Ta ble 960.48. 
(d) Cot ton seed oil. 
(e) Cel lu lose.Cellu Flour, Solka Floc, or equiv a lent. 
(f) Pro tein eval u a tion basal diet. 
trans ported from breed ing col ony  to test lab o ra tory, ac cli ma tion 
pe riod of 3 3 days but <7 should pre cede test. 
Test sam ple  X* 
C. As say Groups 
Cot ton seed oil  8   X    % ether extract  As sem ble groups of 3 10 rats. In ass ay of each mat er ial pro vide 
100  1 group that will rec eive ANRC refe re nce cas ein. One refe re nce 
X    % ash  ca sein group may be used for con cur rent as say of  >1 ass ay mat er ial. 
Salt mix ture USP  5  
100  When ass emb ling of all groups is comp lete, to tal num ber of rats in 
each group must be the same, and av er age weight of rats in any 
Vi ta min mix ture  1  1 group on day be gin ning as say pe riod must not ex ceed by >5 g 
X    % crude fiber  av er age weight of rats in any other group. 
Cel lu lose  1  
100  D. Ass ay Per iod 
X    % moisture  Through out as say pe riod keep each rat in in di vid ual cage and 
Wa ter  5  
100  pro vide with ap pro pri ate as say diet and H2O ad lib it um. Duri ng 
as say  pe riod main tain all con di tions of en vi ron ment as uni form as 
Suc rose or corn starch,
poss ib le with re spect to each of groups be ing comp ared to ANRC 
1.60    100 
to make 100  *X=  ref er ence ca sein. Re cord body weight of each rat on be gin ning day 
% N of test  sample 
of as say pe riod and body weight and food in take of each rat at 
reg u lar in ter vals, not  >7 days, and on 28th day af ter be gin ning of 
All % figu res re fer to test sam ple. Proxi m  ate anal y sis is needed to 
as say pe riod. 
adj ust diet so that all comp ari  sons bet ween test samp les and 
refe re nce mat er ial shall be made with die ts havi ng same cont ent of  E. Cal cu la tions and Tab u la tion  of Re sults 
N, fat, ash, moist ure, and crude fi ber. These sug gested lev els of fat,  Cal cu late av er age 28 day weight gain and pro tein (N    6.25) 
ash, moist ure, and crude fi ber are des ir able when ever prox i mate 
in take per rat for each group. Calc ul ate Pro tein Ef fic iency Ra tio 
anal ys is of product per mits.  (PER) (weight gain/pro tein in take) for each group. De ter mine ra tio   
B. Exp eri m  ent al Ani m  als  100 of PER for each as say group to PER for ANRC ca sein ref er ence 
Labo r at ory rats, males, shall be from same colo ny, and maint ained  group. Tab u late 28 day weight gains, pro tein in take, PER, and ra tio   
dur ing pe riod be fore wean ing upon diet and un der environmental  100 of samp le PER to ANRC Ref er ence Ca sein PER for each ass ay 
con di tions that will pro vide for nor mal de vel op ment in all res pects;  group. Re port pro tein qual ity of samp le as ra tio    100 of samp le PER 
weaned; 3 21 days of age but  28 days of age; range of in di vid ual rat  to ANRC Refe r ence Ca sein PER. 
weights among an i mals used shall be  10 g. When an i mals are  Ref er ences: JAOAC  43, 38(1960); 48, 847(1965). 
a  2005 AOAC IN TER NA TIONAL  Appendix #2. Adjustments required by Association of Official Analytical 
Chemists (AOAC) Official Method 960.48. 
The PER study diets described in AOAC Official Method 960.48 are made up to contain 10% protein (N 
x 6.25), 8% fat, 5% ash, 1% fiber, and 5% moisture.  This is accomplished by use of cottonseed oil, a 
USP salt mixture, cellulose, and water.  Amounts of fat, ash, fiber, and moisture supplied by the test 
sample are accounted for through use of the formulas shown below.  Control and test diets are also 
supplied with 1% each of the same vitamin mixture.  Proximate compositions of the finished diets should 
be the same for each series. 
A worked example of the formulation of the basal diet for a PER study is given below.  The following 
analytical values, obtained by proximate analyses, are used for the calculations: Nitrogen, 13.89%; 
protein (N x 6.25), 86.8%; fat (ether extract), 0.7%; moisture, 7.6%; ash, 1.58%; fiber, 0.47%.  The test 
sample is variously defined as A or X. 
Worked Example of the Formulation of the Basal Diet for a PER Study 
Ingredient  Formula for calculation  Result  Percent in diet 
Test sample (infant  A = (10.0) x 100)/  (1000)/(13.89)(6.25) =  11.52 
formula)  (% protein (N x 6.25)  11.52 
OR  OR 
X = (1.6 x 100)/(% N in  (160)/(13.89)= 11.52 
sample) 
Fat (cottonseed oil)  8-(A x % ether 8-(11.52 x 0.7)/100) = 7.92 
extract)/100
8  0.0806 = 7.919
OR 
8-(X x % ether
extract)/100
Salt mixture (USP)  5-(A x % ash)/100  5  (11.52 x 1.58)/100 =  4.82 
5-0.182 = 4.818
OR 
5-(X x % ash)/100
Cellulose  1-(A x % fiber)/100  1-(11.52 x 1.58)/100 =  0.82 
OR  1-0.182 = 0.818
1-(X x % fiber)/100 
Vitamin mix  None provided  N/A  1 
Water  5-(A x % moisture)/100  5-(11.52 x 7.6)/100 = 5- 4.12 
0.876 = 4.12
OR 
5-(X x % moisture)/100 
Sucrose or  Ingredients: 30.2 %; 
cornstarch, to 100 % 
Sucrose or cornstarch: 
100 - 30.2 = 69.8 % 
37  Appendix #3. Contributions to diets (g/kg) of specific mineral mixtures used at 
listed levels. 
Minerals  USP XIX  USP XIX  Bernhart  AIN- AIN-93G  NRC (1995) 
p.612 p.612 &  76A3  MX3  requirements4
(AOAC)1,2 (AOAC)1 Tomarelli3 
Level of  4%  5%  4%  3.5%  3.5%  - 
addition 
g/kg  g/kg  g/kg  g/kg  g/kg  g/kg 
Ca  6.109  7.636  8.997  5.155  5.0  5.0 
P  3.542  4.428  7.456  3.984  1.992  3.0 
K  4.479  5.5988  2.675  3.602  3.60  3.6 
Na  2.192  2.74  0.763  1.019  1.019  0.5 
Cl  3.381  4.226  0.743  1.571  1.571  0.5 
S  0.771  0.9638  0.501  0.337  0.301  - 
Mg  0.463  0.5788  0.603  0.507  0.502  0.5 
I  0.0242  0.03025  0.0002  0.0002  0.0002  0.00015 
(150 g/kg) 
Fe  0.217  0.2713  0.0373  0.0351  0.036  0.035 
(35 mg/kg) 
Cu  0.0049  0.00613  0.0065  0.0056  00058  0.005 
(5 mg/kg) 
Mn  0.0521  0.06513  0.0464  0.0058  0.0105  0.010 
(10 mg/kg) 
Zn  0.005  0.00625  0.0171  0.0314  0.032  0.012 
(12 mg/kg) 
F  -  -  -  -  0.001  - 
Se  -  -  -  0.00015  0.00015  0.00015 
(150 g/kg) 
Mo  -  -  -  -  -  0.00015 
(150 g/kg) 
Co  0.000228  0.000285  -  -  -  - 
Abbreviations: USP, United States Pharmacopeia; AIN, American Institute of Nutrition; NRC, National 
Research Council. 
1 USP salt mixture XIX (p. 612)((1975).  Corresponding USP XX item (1980) is found in <141> p. 902. 
2 Calculated from 5% level of addition. 
3 Harlan Research Diets_Custom Research Diets.pdf.  Harlan Laboratories, Inc., Indianapolis, IN, 46250; 
http://www.harlan.com, tekladinfo@harlan.com 
4 National Research Council, Subcommittee on Laboratory Animal Nutrition, Committee on Animal Nutrition, 
Board of Agriculture, 1995, Nutrient Requirements of Laboratory Animals, Fourth Revised Edition, National 
Academy Press, Washington, DC.  http://www.nap.edu/catalog/4758.html.  (free download). 
38  Appendix #4. Contributions to diets (units/kg) of specific vitamin mixtures used 
at 1%. 
Vitamin  AOAC Official  AOAC vitamin  AIN-76A  AIN-93G 
method1 mix2  vitamin mix2,3  vitamin 
mix2,3 
Vit. A (dry, stabilized)  20,000 IU  -  -  - 
Dry vit. A palmitate (500,000 IU/g)  -  -  4000 IU  4000 IU 
Vitamin D (dry stabilized)  2000 IU  -  -  - 
Vit. D3 trituration (400,000 IU/g)  -  -  1000 IU  - 
Vit. D3 (cholecalciferol) 500,000  -  -  -  1000 IU 
IU/g 
Vits. A and D powder containing  -  20,000 IU vit A  -  - 
vit. A acetate (500,000 IU/g) and vit. 
D3 (50,000 IU/g)  2000 IU vit D3 
Vit. E (dry stabilized)  100 IU  -  -  - 
Dry vit. E acetate (500 IU/g)  -  100 IU  50 IU  - 
DL-- tocopheryl acetate (500 IU/g) -  -  -  75 IU 
Menadione  5 mg  5 mg  -  - 
Menadione sodium bisulfite  -  -  1.5 mg  - 
complex (62.5% menadione) 
Vit. K (phylloquinone)  -  -  -  0.75 mg 
Choline3  2000 mg (2 g)  -  -  - 
Choline dihydrogen citrate  -  4.8781 g  -  - 
p-aminobenzoic acid 100 mg  100 mg  -  - 
Inositol  100 mg  100 mg  -  - 
Niacin  40 mg  40 mg  30 mg  - 
Nicotinic acid  -  -  -  30 mg 
Ca-D-pantothenate  40 mg  40 mg  16 mg  16 mg 
Riboflavin  8 mg  8 mg  6 mg  6 mg 
Thiamine HCl  5 mg  5 mg  6 mg  6 mg 
Pyridoxine HCl  5 mg  5 mg  7 mg  7 mg 
Folic acid  2 mg  2 mg  2 mg  2 mg 
Biotin  0.4 mg  0.4 mg  0.2 mg  0.2 mg 
Vit. B12  0.03 mg  -  -  - 
Vit. B12 (0.1% trituration in  -  0.03 mg  0.01 mg  0.025 mg 
mannitol) 
Sugar component of vitamin mix  Glucose  Dextrose  Sucrose  Sucrose 
NRC (1995) requirements/kg diet for growth: A (retinol), 0.7 mg (2333 IU); D (cholecalciferol), 0.025 mg; E 
(RRR--tocopherol, 18.0 mg; K (phylloquinone), 1.0 mg;   biotin (d-biotin), 0.2 mg; choline (free 
base), 750 mg; folic acid, 1.0 mg; niacin (nicotinic acid), 15.0 mg; pantothenate (Ca-d-pantothenate), 10.0 
mg; riboflavin, 3.0 mg; thiamin (thiamin-HCl), 4.0 mg; B6 (pyridoxine), 6.0 mg; B12, 50 g. 
1 From formulation in AOAC Official Method 960.48. See Appendix 1. 
2 Harlan Research Diets_ Custom Research Diets.pdf.  Harlan Laboratories, Inc., Indianapolis, IN, 46250; 
http://www.harlan.com., tekladinfo@harlan.com. 
3 The vitamin mixes for diets AIN-76A and AIN-93G do not contain choline or a choline salt.  Choline bitartrate is 
added separately at 2.0 g/kg in diet AIN-76A and at 2.5 g/kg in diet AIN-93G.  See Harlan Research Diets_Custom 
Research Diets.pdf. Harlan Laboratories, Inc., Indianapolis, IN, 46250. 
39  Appendix #5. Compositions of the Association of Official Analytical Chemists 
(AOAC) Official Method 960.48 diet, diets AIN-76A and AIN-93G and 
National Research Council (NRC) Nutrient Requirements of Laboratory 
Animals (1978 and 1995). 
Nutrient  AOAC  AIN-76A  AIN-93G diet  NRC (1978)  NRC (1995) 
  Official  diet    nutrient  nutrient 
  Method      requirements  requirements 
  960.48 diet      for rats  for rats 
           
Sources and  units/100 g  units/100 g  units/100 g  units/100 g  units/100 g 
amounts 
Reference  (1)  (2, 3)  (4)  (5)  (6) 
Protein, g  ANRC  Casein, 20  Casein (> 85%  12  15 
Reference  protein), 20 
Casein 
(10%) (N x 
6.25) 
Fat g  Cottonseed  Corn oil, 5  Soybean oil, 7   5.0  5 
oil, ~8% 
(per 
formula) 
Carbohydrate, g  Sucrose or  Cornstarch,  Corn sugar,  -1  - 
corn starch  15; Sucrose,  39.75; granular 
(per  50  sugar, 10; 
formula)  dextrin, 13.2 
Fiber, g  Cellulose,  Cellulose, 5  Cellulose, 5  -  - 
~1 
Minerals           
Calcium, g  0.763  0.52  0.52  0.5  0.5 
Phosphorus, g  0.443  0.4  0.20  0.4  0.3 
Magnesium, g  0.058  0.05  0.05  0.04  0.05 
Sodium, g  0.274  0.1  0.11  0.05  0.05 
Potassium, g  0.559  0.36  0.38  0.36  0.36 
Chloride, g  0.423  0.16  0.22  0.05  0.05 
Sulfur, g2  0.093  0.033  0.03  0.03  0.03 may be 
beneficial 
Iron, mg  27.1  3.7  5.20  3.5  3.5 
Zinc, mg3   0.624  2.9  3.68  1.2  1.2 
Manganese, mg   6.52  5.9  1.13  5.0  1 
Copper, mg  0. 61  0.6  0.67  0.5  0.5 
Iodine, g  3,025  20  21  15  15 
Selenium, g  -  16  18  10  15 
Cobalt, g  29  -   -  -  - 
Chromium, g  -  200  100  30  - 
Molybdenum,  -  -  15  -  15 
g 
Fluoride, g  -  -  100  100  - 
40  Nutrient  AOAC  AIN-76A  AIN-93G diet  NRC (1978)  NRC (1995) 
  Official  diet    nutrient  nutrient 
  Method      requirements  requirements 
  960.48 diet      for rats  for rats 
           
Sources and  units/100 g  units/100 g  units/100 g  units/100 g  units/100 g 
amounts 
Vitamins 
         
A (retinol), mg 
or IU  2000 IU  400 IU  400 IU  400 IU  230 IU (0.07 
(1 IU = 0.3 g  mg) 
retinol) 
D 
(cholecalciferol),  200 IU  100 IU  100 IU  100 IU  100 IU 
mg or IU  
(1 g  (0.0025 mg) 
cholecalciferol = 
40 IU) 
E (RRR--
tocopherol), mg  10 IU  5 IU  7.5 IU  3 IU  2.7 IU 
or IU 
(1 IU = 0.667  (1.8 mg) 
mg d--
tocopherol) 
K, mg  0.5  0.05  0.09  0.005  0.10 
(menadione)  (menadione)  (phylloquinone)   (phylloquinone)  (phylloquinone) 
or 0.05 
(menadione) 
Biotin (d- 0.04  0.02  0.02  -  0.02 
biotin), mg 
B12, g  3  1  2.5  5  5 
Folic acid, mg  0.2  0.2  0.2  0.10  0.1 
Niacin (nicotinic  4  3  3  2.0  1.5 
acid), mg 
Pantothenate  4  1.6  1.5  0.8  1 
(Ca-d-
pantothenate), 
mg 
B6 (pyridoxine),  0.5  0.7  0.6  0.6  0.6 
mg 
B2 (riboflavin),  0.8  0.6  0.6  0.3  0.3 
mg 
B1 (thiamin),  0.5  0.6  0.5  0.4  0.4 
mg 
Choline, mg  200  200 (choline  250 (choline  100 (choline)  75 (free base) 
(choline)  bitartrate)  bitartrate) 
Inositol, mg  10   -  -  -  - 
p-Aminobenzoic  10   -  -  -  - 
acid, mg 
41  Nutrient  AOAC  AIN-76A  AIN-93G diet  NRC (1978)  NRC (1995) 
  Official  diet    nutrient  nutrient 
  Method      requirements  requirements 
  960.48 diet      for rats  for rats 
           
Sources and  units/100 g  units/100 g  units/100 g  units/100 g  units/100 g 
amounts 
Amino acids           
DL-methionine  -  -  -  -  0.98 
+ cysteine, g 
DL-methionine,  -  0.3  -  -  - 
g 
L-cystine, g  -  -  0.3  -  - 
Antioxidants           
t-butyl- -  -  0.0014  -  - 
hydroquinone, g 
Ethoxyquin, g  -  0.001  -  -  - 
Other           
constituents 
 
1 In otherwise adequate diets, glucose, sucrose, maltose, and fructose support similar levels of 
performance.  In rats fed diets low in protein or water-soluble vitamins, insoluble carbohydrates such as 
starch or dextrin promote more growth than do soluble carbohydrates such as sucrose or glucose (NRC 
(1978) Nutrient Requirements of Laboratory Animals, Third Revised Edition, National Academy Press, 
Washington, DC).  http://nap.nationalacademies.org/20047. 
2 Sulfur: Calculated from composition of mineral salts.  In diet AIN-76-A, the mineral mix contains 52.0 g 
potassium sulfate (18.39% sulfur)/kg mix and is used at 3.5 % of the diet.  Potassium sulfate contributes 0.033 g 
sulfur/100 g diet.  In diet AIN-93G, the mineral mix contains 46.6 g potassium sulfate (18.39% sulfur)/kg mix and 
is used at 3.5% of the diet.  Potassium sulfate contributes 0.0299 g sulfur/kg diet (46.6 x 0.1839 = 8.5697 g 
sulfur/kg salts x 3.5% = 0.0299 g sulfur/100 g).  In the mineral mixtures for diets AIN-76A and AIN-93G, Mg is 
provided as its oxide and Fe is provided as its citrate salt.  Mn, Zn, and Cu are provided as their carbonate salts. 
3 Zn: If rats are housed in galvanized cages, no more than 0.2-0.4 mg Zn/100 g is required (NRC, 1978).  
http://nap.nationalacademies.org/20047. 
References: 
(1) Official Methods of Analysis of AOAC International, 18th edition, 1995, AOAC Official 
Method 960.48 Protein Efficiency Ratio, Rat Bioassay, Section 45.3.04.  Note:  p-
Aminobenzoic acid (PABA) is included in the AOAC PER diets, but not in the AIN diets.  
PABA is an intermediate in the synthesis of folate by bacteria, plants and fungi.  It may have 
been included in the AOAC diets to facilitate synthesis of folate by intestinal bacterial.  Rats do 
not require PABA. 
(2) American Institute of Nutrition, 1977, Report of the American Institute of Nutrition Ad Hoc 
Committee on Standards for Nutritional Studies, Journal of Nutrition 107: 1340-1348 (AIN-
76TM Purified Diet). 
(3) American Institute of Nutrition, 1980, Second report of the ad hoc committee for experimental 
animals.  Journal of Nutrition 110: 1726. 
42  (4) Reeves, P.G., Nielsen, F.H. and Fahey, G.C., Jr. ,1993, AIN-93 Purified Diets for Laboratory 
Rodents: Final Report of the American Institute of Nutrition Ad Hoc Writing Committee on the 
Reformulation of the AIN-76A Rodent Diet, Journal of Nutrition 123: 1939-1951. 
(5) National Research Council, Subcommittee on Laboratory Animal Nutrition, Committee on 
Animal Nutrition, Board of Agriculture, 1978, Nutrient Requirements of Laboratory Animals, 
Third Revised Edition, National Academy Press, Washington, DC.  
http://nap.nationalacademies.org/20047. 
(6) National Research Council, Subcommittee on Laboratory Animal Nutrition, Committee on 
Animal Nutrition, Board of Agriculture, 1995, Nutrient Requirements of Laboratory Animals, 
Fourth Revised Edition, National Academy Press, Washington, DC.  
http://nap.nationalacademies.org/4758. 
43  Appendix #6. Matching the mineral, vitamin, and amino acid compositions of 
PER study test and reference diets. 
One approach to matching the mineral and vitamin compositions of the PER study test and reference diets 
is described below.  The values shown in Table 1 are for illustrative purposes only and should not be 
interpreted as target values.  Columns 1-7 in Table 1 can be completed with the following information: 
Column 1.  List all minerals and vitamins, including their forms and units.  Minerals and vitamins should 
be presented as, for example, Ca (from CaCO3); Vitamin K (as phylloquinone).  All entries should be 
expressed in appropriate units. 
Column 2.  List all mineral and vitamin components in the infant formula in units/100 g or units/kg.  The 
infant formula should be identified by name or associated numbers and a Certificate of Analysis or 
Product Analysis Certificate should be included.  The Certificate of Analysis will usually provide the 
information needed to develop an estimate of the composition of the PER study test diet.  These 
Certificates will often include a list of the test methods by which all nutrients were determined.  Such 
information may indicate the sensitivity and specificity of individual methods. 
Column 3.  Calculate the contribution of the infant formula to the PER study test diet based on the use 
rate or incorporation rate (e.g., 85%, 90%) of the infant formula in the test diet.  Review the NRC (1995) 
nutrient requirements for rats (Column 7) to determine the adequacy of the composition of the test diet at 
this stage. 
Column 4.  Identify minerals or vitamins that do not meet the NRC (1995) specifications.  The NRC 
specifications are minimal and do not include a margin of safety.  Calculate the amounts of minerals and 
vitamins that need to be added to meet the NRC (1995) levels with a margin of safety (e.g., ~ 10%).  
Identify those that may be near or at a potentially toxic level based on information provided by the NRC 
(1995).  Several worked examples are shown in the Table.  Formulate mineral or vitamin mixtures that 
are needed to bring deficient levels, if any, in Column 3 to NRC (1995) levels.  List the contributions to 
the test diet from these mineral or vitamin mixtures in Column 4. 
Column 5.  Add Columns 3 and 4 to obtain the estimated final composition of the PER study test diet.  
Review the composition against the NRC (1995) nutrient specifications for rats to confirm the adequacy 
of the estimates. 
Column 6.  The composition of the PER study reference diet is developed from the estimates in Column 
5.  For each mineral and vitamin, calculate a range that is 20% around the estimated value in Column 5.  
Compare the low and high ends of the calculated range against NRC (1995) values for nutrient.  
Formulate vitamin and mineral mixtures needed for the reference diet and list their contributions in 
column 6.  All estimates should fall within the ranges identified in Column 5 (that is, the estimates for the 
reference diet should fall within 20% of the final estimates for the test diet in Column 5).  Compare all 
estimates with NRC (1995) levels and adjust as needed to reduce the possibility of potentially deficient or 
toxic levels of specific minerals or vitamins. 
Fiber (see Section IV.B.1.g of the guidance):  Infant formulas frequently contain non-digestible 
carbohydrates such as fructo-oligosaccharides (FOS) or galacto-oligosaccharides (GOS).  Our current 
thinking is that it is not necessary to add cellulose to the infant formula test diet or to its matched casein 
control diet when the infant formula includes non-digestible carbohydrates such as FOS and/or GOS.  
When FOS or GOS are present in the infant formula, then they should be included in the matched casein 
control diet.  Due to our uncertainty as to the outcome of PER studies conducted in completely fiber-free 
diets, we further recommend that 1% cellulose be added to the test and matched casein control diet when 
the infant formula does not contain a source of non-digestible carbohydrate. 
44  Vitamin E and Polyunsaturated Fatty Acids (PUFA) (see Section IV.B.1.c of the guidance):  The 
need for vitamin E increases with an increase in consumption of PUFA and with the degree of 
unsaturation of dietary PUFAs.  The following adjustment takes this into consideration:  The vitamin E 
and PUFA contents of the PER study test and reference diets can be estimated from the Certificates of 
Analysis or other information.  The ratios of vitamin E:PUFA expressed as mg vitamin E/g PUFA can be 
calculated.  The ratio of 0.48  0.28 mg of vitamin E per gram of PUFA is used as a general guideline for 
the relationship between these nutrients.  Modifications in the concentration of vitamin E in the reference 
diet can be made as needed.  See the Table below for an example. 
Inorganic Sulfur:  While there is uncertainty regarding an appropriate concentration of inorganic sulfur 
to add to the PER study casein reference control diet, we are recommending that the concentration of 
inorganic sulfur in the control diet be adjusted to provide the inorganic sulfur content set forth in the 
AOAC Method (i.e., 0.964 g/kg diet). 
Sulfur Amino Acids (methionine, cystine) (see Section IV.B.1.h of the guidance) (Table 2): Diets 
containing 8-10% protein from casein are limiting in sulfur amino acids methionine and cystine.  
Sponsors may want to consider matching the concentrations of (methionine + cystine) in the PER study 
test and reference casein diets if there is concern about a potential failure of the casein reference group 
because of low concentrations of cystine in casein.  This matching is optional and can be performed at the 
discretion of the sponsor.  Sponsors who choose to prepare the (methionine + cystine)-matched diet 
described below may want to include it as a second casein reference control group in their PER study. 
The matching may be performed as follows:  The infant formula and the casein for the control diet should 
be analyzed for their methionine and cystine content prior to diet preparation.  The concentrations of 
(methionine + cystine) should be calculated from the results of these analyses.  If the concentration of 
cystine estimated to be present in the control diet is <80% of the value for cystine in the test diet, then 
cystine can be added to the control diet so that the concentrations of (methionine + cystine) are similar in 
both diets.  The final concentration of (methionine + cystine) in the control diet should be 20% of the 
value for (methionine + cystine) in the test diet.  Addition of methionine alone is not an appropriate 
remedy for a deficit of cystine in the casein control diet.  See the Table below for an example. 
In cases in which the sum of (methionine + cystine) in the test diet is less than the sum of (methionine + 
cystine) in the control diet, and the amount of cystine in the control diet is < 80% of the value for cystine 
in the test diet, then cystine can be added to the control diet so that the concentrations of cystine are the 
same in both the control and reference diets. 
Use of NRC (1995) Nutrient Requirements of Laboratory Animals (Reference 1):  The NRC (1995) 
values are expressed on an as-fed basis for diets containing 10% moisture and 3.8-4.1 kcal metabolizable 
energy/g and should be adjusted for diets of differing moisture and energy concentrations.  In addition, 
the NRC (1995) levels for growth are for diets containing 15% protein and 5% fat.  Unless otherwise 
specified, the listed nutrient levels represent minimal values and do not include a margin of safety. 
We consider that most nutrient levels would be significantly lower for the 10% PER study diets than the 
NRC (1995) values for 15% protein diets, but we are not able to determine the reductions that might be 
appropriate for each nutrient.  While we are unable to provide specific guidance in this area, we consider 
that the NRC (1995) values should be used to identify and correct a PER study diet that may be deficient 
in a required mineral or vitamin.  The same consideration applies to identifying and correcting a PER 
study diet that may be formulated to contain a potentially toxic level of a specific mineral or vitamin. 
The values shown below are for illustrative purposes only and should not be interpreted as target values.  
The composition of each infant formula must be evaluated independently in order to determine how to 
formulate appropriate PER study test and reference diets. 
45  Table 1. Example of an approach for matching mineral and vitamin compositions of PER study test 
and reference diets. 
Column 1  Column 2  Column 3  Column 4  Column 5  Column 6  Column 7 
Mineral or  Composition  Contribution  Additions  Test diet,  Reference  NRC (1995) 
vitamin  of infant  of infant  to test diet  final  diet = test  nutrient 
formula,  formula to  diet  requirements 
test diet 
 20% 
Rate of use 
(%), 90 
Sources and  units/100 g  units/100 g  units/100 g  units/100 g  units/100 g  units/100 g 
amounts 
Minerals 
Calcium  0.50  0.45  0.05  0.50  0.40-0.60  0.5 
(CaCO ), g 
3
Phosphorus, g  0.40  0.36  none  0.36  0.29-0.43  0.3 
Magnesium, g  0.06  0.054  none  0.054  0.043-0.065  0.05 
Sodium, g  0.22  0.20  none  0.20  0.16-0.24  0.05 
Potassium, g  0.67  0.60  none  0.60  0.48-0.72  0.36 
Chloride, g  0.33  0.30  none  0.30  0.24-0.36  0.05 
Sulfur, g  -  -  -  -  -  0.03 
Iron, mg  20.00  18  none  18  14.4-21.6  3.5 
Zinc, mg  0.90  0.81  0.39  1.20  0.96-1.44  1.2 
Manganese, mg  1.67  1.5  none  1.5  1.19-1.80  1 
Copper, mg  0.67  0.60  none  0.60  0.48-0.72  0.5 
Iodine, g  88.90  80  none  80  64-96 15 
Selenium, g  22.20  20  none  20  16-24 15 
Cobalt, g  -  12  12  -  Not required 
Chromium, g  -  -  -  -  Not required 
Molybdenum,  16.70  15  none  15  12-18 15 
g 
Fluoride, g  -  -  -  -  -  Not required 
Vitamins 
A (retinol), mg  5,000 IU  4500 IU  none  4500 IU  3600-5400  230 IU (0.07 
or IU; 1 IU = 0.3  IU  mg) 
g retinol 
-carotene  -  -  -  -  -  - 
D3  680 IU  612 IU  none  612 IU  490-734 IU 100 IU 
(cholecalciferol),  (0.0025 mg) 
mg or IU; 1 g 
D3= 40 IU 
 
E (RRR-- 40 IU  36 IU  none  36 IU  29-43 IU 2.7 IU (1.8 
tocopherol), mg  mg) 
or IU; 1 IU = 
0.667 mg d--
tocopherol 
K  0.08  0.072  0.028  0.10  0.08-0.12  0.10 
(phylloquinone), 
mg 
46  Column 1  Column 2  Column 3  Column 4  Column 5  Column 6  Column 7 
Mineral or  Composition  Contribution  Additions  Test diet,  Reference  NRC (1995) 
vitamin  of infant  of infant  to test diet  final  diet = test  nutrient 
formula,  formula to  diet  requirements 
test diet 
 20% 
Rate of use 
(%), 90 
Sources and  units/100 g  units/100 g  units/100 g  units/100 g  units/100 g  units/100 g 
amounts 
Biotin (d-biotin),  0.03  0.027  none  0.027  0.022-0.032  0.02 
mg 
B12, g  5.0  4.5  0.50  5.0  4.00-6.00  5 
Folic acid, mg  0.22  0.20  none  0.20  0.16-0.24  0.1 
Niacin (nicotinic  7.78  7.0  none  7.0  5.60-8.40  1.5 
acid), mg 
Pantothenate  5.00  4.5  none  4.5  -  1 
(Ca-d-
pantothenate), 
mg 
B6 (pyridoxine),  0.90  0.81  none  0.81  0.65-0.97  0.6 
mg 
B2 (riboflavin),  1.11  1.0  none  1.0  0.80-1.20  0.3 
mg 
B1 (thiamin),  1.00  0.90  none  0.90  0.72-1.08  0.4 
mg 
Choline, mg  3.89  350  none  350  280-420 75 (free 
base) 
Inositol, mg  33.3  30  none  30  24-36 Not required 
p-Aminobenzoic -  -  -  -  -  Not required 
acid, mg
Vitamin C, mg  166.67  150  150  120-180 Not required 
Fiber  See  -  -  -  -  -
Narrative 
Section 
above 
47  Column 1  Column 2  Column 3  Column 4  Column 5  Column 6  Column 7 
Mineral or  Composition  Contribution  Additions  Test diet,  Reference  NRC (1995) 
vitamin  of infant  of infant  to test diet  final  diet = test  nutrient 
formula,  formula to  diet  requirements 
test diet 
 20% 
Rate of use 
(%), 90 
Sources and  units/100 g  units/100 g  units/100 g  units/100 g  units/100 g  units/100 g 
amounts 
Vitamin E, mg  5.0  4.5  none  4.5  3.6-5.4  The mean 
PUFA, g  7.0  6.3  6.3  5.04-7.56  value of 0.48 
mg vitamin 
E/g PUFA 
(or 48 mg 
vitamin 
E/100 g 
Ratio of mg  0.71 mg/g  0.71 mg/g  Estimated  PUFA) is a 
vitamin E/g  range of  general 
PUFA  ratios:  guideline for 
3.6/5.04=0.7 the minimum 
1  ratio of the 
3.6/7.56=0.4 two 
8  nutrients. 
5.4/5.04=1.0
7 
5.4/7.56=0.7
1 
~0.48 - 1.07 
mg/g 
Ratio of mg  71 mg/100 g  71 mg/100  ~48-107 
vitamin E/100 g  g  mg/100g 
PUFA 
none:  The contribution of the infant formula to the test diet meets or exceeds the NRC (1995) 
(Reference 1) specifications.  No addition is needed. 
48  Table 2. Optional inclusion of a second reference casein control group with diet matched to cystine 
+ methionine content of the infant formula-based test diet. 
Amino acid  Composition  Contribution  Addition to  Test  Addition to  Variability 
of infant  of infant  test diet  diet  reference  in 
formula  formula to    final  diet  reference 
  test diet        diet 
           
  Rate of use         
  (%), 90         
           
g/100 g  g/100 g  g/100 g  g/100 g  g/100 g  (%) 
Cystine  0.243  0.219  N/A  0.219  0.037+0.084  Value for 
Methionine  0.268  0.241  N/A  0.241  0.339  SAA in the 
          reference 
          diet should 
Sum  0.511  0.460  0.460  0.460  be  20% of 
the value 
for SAA in 
the test diet. 
Cystine/methionine:  In this example, assume that casein provides per 100 g: 0.37 g cystine and 3.39 g 
methionine for a total of 3.76 g/100 g sulfur amino acids.  Cystine provides 9.8% of the SAA in casein 
(0.037/0.376) x 100 = 9.8%).  When used at 10% of the reference diet, casein provides per 100 g of diet: 
0.037 g cystine and 0.339 g methionine.  In this example, cystine provided by casein in the reference diet 
is 16.9% of the amount of cystine provided in the infant formula diet ((0.037/0.219) x 100 = 16.9%). 
The amount of cystine to be added to the casein reference diet is calculated as follows (g/100 g): (sum of 
cystine + methionine in test diet) less (sum of cystine and methionine in casein reference diet) = 0.460  
0.037  0.3339 = 0.084 g cystine/100 g to be added to the reference diet.  When this addition is 
accomplished, the test diet and reference diet have the same concentrations of SAA. An example of this 
approach is found in Hoskin, 2022;  https://doi.org/10.1093/jaoacint/qsac110. 
Cystine contributes 47.6% (0.219/0.046 = 0.476 x 100 = 47.6%) of the total SAA in the infant formula 
test diet and 26.3% ((0.037 + 0.084)/0.460 = 0.263 x 100 = 26.3%) of the total SAA in the casein 
reference diet. 
N/A: Not applicable. 
Reference: 
(1) Nutrient Requirements of Laboratory Animals, Fourth Revised Edition, 1995, Subcommittee on 
Laboratory Animal Nutrition, National Research Council, National Academies Press, 
Washington, DC, Chapter 2, pages 11-79.  http://nap.nationalacademies.org/4758. 
49