Dietary Supplements: General Chapters Information
Dietary Supplements: General Chapters Information
Dietary Supplements: General Chapters Information
Dietary Supplements
INTRODUCTION
This chapter provides tests for the estimation of the number of viable aerobic microorganisms present in nutritional supple-
ments of all kinds, from raw materials to the finished forms. Alternative methods may be substituted for the tests, provided
that they have been properly validated as giving equivalent or better results. In preparing for and in applying the tests, observe
aseptic precautions in handling the specimens. The term “growth” is used in a special sense herein, i.e., to designate the pres-
ence and presumed proliferation of viable microorganisms.
PREPARATORY TESTING
The validity of the results of the tests set forth in this chapter rests largely upon the adequacy of a demonstration that the
test specimens to which they are applied do not, of themselves, inhibit the multiplication, under the test conditions, of micro-
organisms that may be present. Therefore, preparatory to conducting the tests on a regular basis and as circumstances require
subsequently, inoculate diluted specimens of the material to be tested with separate viable cultures of the challenge microor-
ganisms.
For the Soybean–Casein Digest Agar Medium used for Total Aerobic Microbial Count, inoculate duplicate plates with 25–250
cfu of Staphylococcus aureus (ATCC1 No. 6538), Escherichia coli (ATCC No. 8739), and Bacillus subtilis (ATCC No. 6633) to dem-
onstrate a greater than 70% bioburden recovery in comparison to a control medium. For the Sabouraud Dextrose Agar Medium
used for Total Combined Molds and Yeasts Count, inoculate duplicate plates with 25–250 cfu of Candida albicans (ATCC No.
10231) and Aspergillus brasiliensis (ATCC No. 16404) to demonstrate a greater than 70% bioburden recovery in comparison to
a control medium. For Enterobacterial Count (Bile-Tolerant Gram-Negative Bacteria), appropriate dilutions of Escherichia coli
(ATCC No. 8739) and Salmonella typhimurium (ATCC No. 13311) are used. Failure of the organism(s) to grow in the relevant
medium invalidates that portion of the examination and necessitates a modification of the procedure by (1) an increase in the
volume of diluent, the quantity of test material remaining the same, or by (2) the incorporation of a sufficient quantity of suita-
ble inactivating agent(s) in the diluents, or by (3) an appropriate combination of modifications to (1) and (2) so as to permit
growth of the inoculum.
The following are examples of ingredients and their concentrations that may be added to the culture medium to neutralize
inhibitory substances present in the sample: soy lecithin, 0.5%; and polysorbate 20, 4.0%. Alternatively, repeat the test as de-
scribed in the preceding paragraph, using Fluid Casein Digest–Soy Lecithin–Polysorbate 20 Medium to demonstrate neutralization
of preservatives or other antimicrobial agents in the test material. Where inhibitory substances are contained in the product
and the latter is soluble, a suitable, validated adaptation of a procedure set forth under Procedure using the Membrane Filtration
Method may be used.
If, in spite of the incorporation of suitable inactivating agents and a substantial increase in the volume of diluent, it is still not
possible to recover the viable cultures described above, and where the article is not suitable for the employment of membrane
filtration, it can be assumed that the failure to isolate the inoculated organism is attributable to the bactericidal or bacteriostat-
ic activity of such magnitude that treatments are not able to remove the activity. This information serves to indicate that the
article is not likely to allow proliferation or contamination with the given species of microorganism. Monitoring should be con-
tinued in order to determine the inhibitory range and bactericidal activity of the article.
1 Available from ATCC, 10801 University Boulevard, Manassas, VA 20110-2209. Equivalent microorganisms, provided that they are from a national collection re-
pository, can be used in lieu of ATCC strains. However, the viable microorganisms used in the test must not be more than five passages removed from the original
ATCC or national collection culture.
Official text. Reprinted from First Supplement to USP38-NF33.
790 á2021ñ Microbial Enumeration Tests / Dietary Supplements DSC
Culture media may be prepared as follows, or dehydrated culture media may be used provided that, when reconstituted as
directed by the manufacturer or distributor, they have similar ingredients and/or yield media comparable to those obtained
from the formulas given herein.
In preparing media by the formulas set forth herein, dissolve the soluble solids in the water, using heat if necessary to effect
complete solution, and add solutions of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the desired pH
in the medium when it is ready for use. Determine the pH at 25 ± 2°.
Where agar is called for in a formula, use agar that has a moisture content of NMT 15%. Where water is called for in a for-
mula, use Purified Water.
Prepare a stock solution by dissolving 34 g of monobasic potassium phosphate in about 500 mL of water contained in a
1000-mL volumetric flask. Adjust to a pH of 7.2 ± 0.1 by the addition of sodium hydroxide TS (about 175 mL), add water to
volume, and mix. Dispense and sterilize. Store under refrigeration. For use, dilute the stock solution with water in the ratio of
1–800, dispense as desired, and sterilize.
Media
Prepare media for the tests as described below. Alternatively, dehydrated formulations may be used provided that, when
reconstituted as directed by the manufacturer or distributor, they meet the requirements of Growth Promotion Testing. Unless
otherwise indicated elsewhere in this chapter, media are sterilized in autoclaves using a validated process. The exposure time
within the autoclave at 121° will depend on the volume of media to be sterilized. Thus, for example, a 500-mL volume would
need to be autoclaved using a temperature and time relationship that will ensure that the medium has attained at least an F0
of 12–15 in the sterilization process. However, the appropriate time and temperature duration for sterilizing prepared media at
any given volume should be confirmed by a thermal penetration study using a thermocouple or thermoprobe placed within
the liquid medium.
Dissolve Pancreatic Digest of Casein and Soy Lecithin in 960 mL of water, heating in a water bath at 48°–50° for about 30 min
to effect solution. Add 40 mL of Polysorbate 20. Mix, dispense as desired, and sterilize.
Dissolve the solids in the water, heating slightly to effect a solution. Cool the solution to room temperature, and adjust the
pH with 1 N sodium hydroxide so that after sterilization it will have a pH of 7.3 ± 0.2. Filter, if necessary, and dispense into
suitable containers. Sterilize at a temperature and time relationship that will ensure that the medium has attained at least an F0
of 12–15 in the sterilization process, or by a validated filtration process.
Dextrose 40.0 g
Mixture of Peptic Digest of Animal Tissue and Pancreatic Digest of Casein (1:1) 10.0 g
Agar 15.0 g
Water 1000 mL
Adjust the pH so that it is 7.4 ± 0.2 after heating. Heat to boiling, but do not heat in an autoclave. Pour onto plates.
Suspend the solids in water, and heat to boiling for 1–2 min. Transfer 120-mL portions to 250-mL volumetric flasks or 9-mL
portions to test tubes, all being capped with cotton plugs or loose-fitting caps. Heat on a steam bath for 30 min. Adjust the pH
so that it is 7.2 ± 0.2 after heating.
Each lot of dehydrated medium bearing the manufacturer's identifying number or each lot of medium prepared from basic
ingredients must be tested for its growth-promoting qualities. Cultures of Staphylococcus aureus (ATCC No. 6538), Escherichia
coli (ATCC No. 8739), Bacillus subtilis (ATCC No. 6633), Candida albicans (ATCC No. 10231), and Aspergillus brasiliensis (ATCC
No. 16404) are used. A 10–3 dilution of a 24-hour broth culture of the microorganism to the first dilution (in pH 7.2 Phosphate
Buffer or Fluid Soybean–Casein Digest Medium) may be used as the inocula. Serially streak plates of the media with the appropri-
ate inocula to obtain isolated colonies to demonstrate the growth-promotion qualities of the Soybean–Casein Digest Agar and
Sabouraud Dextrose Agar media. Inoculate the Fluid Soybean–Casein Digest Medium and Mossel–Enterobacteriaceae Enrichment
Broth with 10–100 cfu of the appropriate challenge organisms to demonstrate their growth-promotion qualities.
SAMPLING
Provide 10-mL or 10-g specimens for the tests called for in the individual monograph.
PROCEDURE
Prepare the specimen to be tested by a treatment that is appropriate to its physical characteristics and that does not alter
the number and kind of microorganisms originally present, in order to obtain a solution or suspension of all or part of it in a
form suitable for the test procedure(s) to be carried out.
For a solid that dissolves to an appreciable extent but not completely, reduce the substance to a moderately fine powder,
suspend it in the vehicle specified, and proceed as directed under Total Aerobic Microbial Count.
For a fluid specimen that consists of a true solution, or a suspension in water or a hydroalcoholic vehicle containing less than
30% of alcohol, and for a solid that dissolves readily and practically completely in 90 mL of pH 7.2 Phosphate Buffer or the
media specified, proceed as directed under Total Aerobic Microbial Count.
For water-immiscible products, prepare a suspension with the aid of a minimal quantity of a suitable, sterile emulsifying
agent (such as one of the polysorbates), using a mechanical blender and warming to a temperature not exceeding 45°, if nec-
essary, and proceed with the suspension as directed under Total Aerobic Microbial Count.
For specimens that are freely soluble, use the Membrane Filtration Method or Plate Method. For specimens that are sufficiently
soluble or translucent to permit use of the Plate Method, use that method; otherwise, use the Multiple-Tube Method. With either
method, first dissolve or suspend 10.0 g of the specimen if it is a solid, or 10 mL, accurately measured, if the specimen is a
liquid, in pH 7.2 Phosphate Buffer, Fluid Soybean–Casein Digest Medium, or Fluid Casein Digest–Soy Lecithin–Polysorbate 20 Medi-
um to make 100 mL. For viscous specimens that cannot be pipeted at this initial 1:10 dilution, dilute the specimen until a
suspension is obtained, i.e., 1:50 or 1:100, etc., that can be pipeted. Perform the test for absence of inhibitory (antimicrobial)
properties as described under Preparatory Testing before the determination of Total Aerobic Microbial Count. Add the specimen
to the medium NMT 1 h after preparing the appropriate dilutions for inoculation.
Dilute the fluid further, if necessary, so that 1 mL will be expected to yield 30–300 colonies. Pipet 1 mL of the final dilution
into 5–10 mL of pH 7.2 Phosphate Buffer, Fluid Soybean–Casein Digest Medium, or Fluid Casein Digest–Soy Lecithin–Polysorbate
20 Medium. Wash each membrane with an appropriate amount of one of the above diluents. Transfer each membrane to a
Petri dish containing Soybean–Casein Digest–Agar Medium, previously solidified at room temperature. Incubate the plates at a
temperature 30°–35° for 48–72 h. Following incubation, examine the plates for growth, count the number of colonies, and
express the average for the two plates in terms of the number of microorganisms per g or per mL of specimen. If no microbial
colonies are recovered from the dishes representing the initial 1:10 dilution of the specimen, express the results as “less than
10 microorganisms per g or per mL of specimen”.
PLATE METHOD
Dilute the fluid further, if necessary, so that 1 mL will be expected to yield 30–300 colonies. Pipet 1 mL of the final dilution
onto each of two sterile Petri dishes. Promptly add to each dish 15–20 mL of Soybean–Casein Digest–Agar Medium, previously
melted and cooled to about 45°. Cover the Petri dishes, mix the sample with agar by gently tilting or rotating the dishes, and
allow the contents to solidify at room temperature. Invert the Petri dishes and incubate for 48–72 h. Following incubation,
examine the plates for growth, count the number of colonies, and express the average for the two plates in terms of the num-
ber of microorganisms per g or per mL of specimen. If no microbial colonies are recovered from the dishes representing the
initial 1:10 dilution of the specimen, express the results as “less than 10 microorganisms per g or per mL of specimen”.
MULTIPLE-TUBE METHOD
Into each of 14 test tubes of similar size, place 9.0 mL of sterile Fluid Soybean–Casein Digest Medium. Arrange 12 of the tubes
in four sets of three tubes each. Put aside one set of three tubes to serve as the controls. Into each of three tubes of one set
(“100”) and into a fourth tube (A) pipet 1 mL of the solution or suspension of the specimen, and mix. Pipet 1 mL from tube A
into the one remaining tube (B), not included in a set, and mix. These two tubes contain 100 mg or 100 mL and 10 mg or 10
mL of the specimen, respectively. Into each of the second set (“10”) of three tubes pipet 1 mL from tube A, and into each tube
of the third set (“1”) pipet 1 mL from tube B. Discard the unused contents of tubes A and B. Close well, and incubate all of the
tubes. Following incubation, examine the tubes for growth: the three control tubes remain clear, and the observations in the
tubes containing the specimen, when interpreted by reference to Table 1, indicate the most probable number of microorgan-
isms per g or per mL.
Procedure—Proceed as directed for Membrane Filtration Method or Plate Method under Total Aerobic Microbial Count, except
to use the same amount of Sabouraud Dextrose–Agar Medium instead of Soybean–Casein Digest–Agar Medium and to incubate
the plates for 5–7 days at 20°–25°.
Retest—For the purpose of confirming a doubtful result by any of the procedures outlined in the foregoing tests following
their application to a 10-g specimen, a retest on an additional 10-g specimen from the original sample and a 10-g specimen
from the new sample of the nutritional supplement may be conducted. Proceed as directed under Procedure.
Dissolve or suspend the sample in a sufficient volume of pH 7.2 Phosphate Buffer or Fluid Soybean–Casein Digest Medium and
dilute with Fluid Soybean–Casein Digest Medium to 100 mL. Pre-incubate for 2–5 h at 20°–25° in soybean–casein digest broth
diluent; inoculate suitable quantities of Mossel–Enterobacteriaceae Enrichment Broth to contain 0.1, 0.01, or 0.001 g or mL of
the product. Incubate at 30°–35° for 24–48 h. Subculture onto a plate of Violet-Red Bile Agar with Glucose and Lactose, and
incubate at 30°–35° for 18–24 h. Growth of well developed, generally red or reddish, colonies of Gram-negative bacteria re-
veal the presence of enterobacteria. Determine the most probable number of microorganisms per g or per mL by reference to
Table 2.
Table 2. Most Probable Enterobacterial Count
Observed Presence of Enterobacteria
Number of g or mL of specimen per tube Most Probable Number of Enterobacteria
0.1 0.01 0.001 per g or per mL
+ + + More than 103
INTRODUCTION
Good manufacturing practices require that objectionable organisms be absent from nonsterile nutritional and dietary prod-
ucts. A microorganism can be considered objectionable if it represents a potential health hazard to the user who is using the
product as directed, or if it is capable of growing in the product. Objectionable microorganisms are defined as contaminants
that, depending on the microbial species, number of organisms, dosage form, intended use, and patient population, would
adversely affect product safety. Additionally, microorganisms may be deemed objectionable if they adversely affect product
stability or if they may damage the integrity of the container closure system.
This chapter describes the testing of nutritional and dietary articles for specified microorganisms, which are specified in the
individual monographs or whose absence is recommended by the guidance under Microbiological Attributes of Nonsterile Nutri-
tional and Dietary Supplements á2023ñ. When objectionable microorganisms are not specified in the individual monograph, it is
the manufacturers' responsibility to determine which microorganisms in their products are objectionable. It is not intended
that all nonsterile nutritional and dietary articles be tested for the absence of all of the microorganisms mentioned in this chap-
ter, nor is the testing of relevant microorganisms restricted to those presented in this chapter.
Alternative microbiological, physicochemical, and biotechnological methods, including automated methods, may be substi-
tuted for these tests, provided they have been validated as being equivalent in their suitability for determining compliance.
General Considerations
See Buffer Solution and Media under Microbial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ. The appropri-
ateness of each medium for the intended purpose is to be assessed. Control sets of Fluid Soybean–Casein Digest Medium for
Preparatory Testing are also used to assess the appropriateness of these media in the growth promotion of the specified micro-
organisms. For other media, streak agar plates to obtain isolated colonies of appropriate microorganisms, and inoculate the
fluid media with the appropriate microorganisms at a final concentration of less than 100 cfu per mL. Observe the growth to
establish the appropriateness of the media.
Buffer
Buffer Stock Solution and pH 7.2 Phosphate Buffer—Proceed as directed under Microbial Enumeration Tests—Nutritional
and Dietary Supplements á2021ñ.
Media
Prepare as directed under Microbial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ.
MANNITOL–SALT–AGAR MEDIUM
Mix, then heat with frequent agitation, and boil for 1 minute to effect solution.
pH after sterilization: 7.4 ± 0.2.
Heat to boiling. Do not autoclave; use the same day. Immediately before use, add a solution prepared by dissolving 5 g of
potassium iodide and 6 g of iodine in 20 mL of water. Then add 10 mL of a solution of brilliant green (1 in 1000), and mix. Do
not heat after adding the brilliant green solution.
Boil for 1 minute. Sterilize just prior to use, melt, pour into Petri dishes, and allow to cool.
pH after sterilization: 6.9 ± 0.2
XYLOSE–LYSINE–DESOXYCHOLATE–AGAR MEDIUM
Xylose 3.5 g
L-Lysine 5.0 g
Lactose 7.5 g
Sucrose 7.5 g
Sodium Chloride 5.0 g
Yeast Extract 3.0 g
Phenol Red 80 mg
Agar 13.5 g
Sodium Desoxycholate (as Bile Salts) 2.5 g
Sodium Thiosulfate 6.8 g
Ferric Ammonium Citrate 800 mg
Water 1000 mL
Heat, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to a water bath maintained at
about 50°, and pour into Petri plates as soon as the Medium has cooled.
Final pH: 7.4 ± 0.2.
Mix, and allow to stand for 10 minutes. Heat gently, and allow to boil for a few seconds to dissolve the agar. Do not steri-
lize. Cool to 60°, and pour into Petri dishes.
Final pH: 7.5 ± 0.2.
Agar 15.0 g
Lactose 10.0 g
Eosin Y 400 mg
Methylene Blue 65 mg
Water 1000 mL
Dissolve pancreatic digest of gelatin, dibasic potassium phosphate, and agar in water, with warming, and allow to cool. Just
prior to use, liquefy the gelled agar solution, and add the remaining ingredients, as solutions, in the following amounts: for
each 100 mL of the liquefied agar solution, add 5 mL of lactose solution (1 in 5), 2 mL of the eosin Y solution (1 in 50), and 2
mL of methylene blue solution (1 in 300). Mix. The finished Medium may not be clear.
pH after sterilization: 7.1 ± 0.2.
Heat with frequent agitation, and boil for 1 minute. Sterilize, cool to between 45° and 50°, and add 10 mL of sterile potassi-
um tellurite solution (1 in 100) and 50 mL of egg yolk emulsion prepared as follows. Disinfect the surface of whole-shell eggs,
aseptically crack the eggs, transfer intact yolks to a sterile graduated cylinder, add sterile saline TS to obtain a 3 to 7 ratio of
egg yolk to saline, add to a sterile blender cup, and mix at high speed for 5 seconds. Mix all ingredients well but gently, and
pour into plates.
pH after sterilization: 6.8 ± 0.2.
Boil for 1 minute. Sterilize, cool to between 45° and 50°, and add 20 mL of sterile potassium tellurite solution (1 in 100).
pH after sterilization: 7.2 ± 0.2.
Mix, and heat to effect solution. Then heat in flowing stream for 15 minutes. Do not sterilize.
Final pH: 7.0 ± 0.2.
Dissolve agar in water by heating to boiling, while stirring continuously. Adjust the pH if necessary, and sterilize.
pH after sterilization: 6.8 ± 0.2.
COLUMBIA AGAR
Dissolve agar in water by heating to boiling and with continuous stirring. If necessary, adjust the pH. Sterilize, and allow to
cool to 45° to 50°. Add, when necessary, gentamicin sulfate, equivalent to about 20 mg of gentamicin base, and pour into
Petri dishes.
Pre-reduction of the medium is recommended.
pH after sterilization: 7.3 ± 0.2.
Dissolve, warming slightly. Sterilize in an autoclave using a validated cycle, at a temperature not exceeding 115°.
The pH is 5.2 ± 0.2 at 25° after heating and autoclaving.
PREPARATORY TESTING
Proceed as directed for Preparatory Testing under Microbial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ.
For enrichment broth, selective media, and differential media use an inoculating loop to transfer the inoculum of each test
organism to the plated or liquid media being tested. If a plated medium is being tested, streak the surface of plate with the
loop in four directions to obtain a pattern of isolated colonies. Incubate the media, and examine the plated or liquid media for
the characteristic growth of the inocula (See Tables 1, 2, 3, and 4).
SAMPLING
Proceed as directed for Sampling under Microbial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ.
TEST PROCEDURES
Test Preparation—Prepare as directed for Sampling. Transfer to a suitable container with 100 mL of Fluid Soybean–Casein
Digest Medium (FSCD). Mix by shaking gently. [NOTE—On the basis of results for Preparatory Testing, modify the Test Prepara-
tion as appropriate.]
Incubate at 30° to 35° for 18 to 24 hours. Streak a loopful from FSCD onto the surface of one or more of the following me-
dia: Vogel–Johnson Agar Medium (VJ Agar), Mannitol–Salt–Agar Medium (MS-Agar), and Baird-Parker Agar Medium (BP Agar). Cov-
er the Petri plates, invert them, and incubate at 30° to 35° for 24 to 48 hours.
Examine the plates of VJ Agar, MS-Agar, and/or BP Agar, and interpret the results with reference to Table 1: if no plate con-
tains colonies having the characteristics described, the test specimen meets the requirement for the absence of Staphylococcus
aureus. If characteristic colonies are present, perform coagulase test as follows. Transfer representative colonies to separate
tubes containing 0.5 mL of rabbit plasma, horse plasma, or any other mammalian plasma. Incubate in a water bath at 37°.
Examine for coagulation after 3 hours of incubation and at suitable intervals up to 24 hours. Comparing with positive and neg-
ative controls, the absence of a coagulase reaction indicates the absence of Staphylococcus aureus in the tested article.
Table 1. Characteristics of Staphylococcus aureus on Specified Agar Media
Agar Medium Colonial Morphology Gram Stain
Vogel–Johnson Black surrounded by yellow zone (+), cocci
Mannitol–Salt Yellow colonies with yellow zone (+), cocci
Black, shiny surrounded by
Baird–Parker 2–5-mm clear zones (+), cocci
Incubate at 30° to 35° for 18 to 24 hours. From FSCD, pipet a 1-mL aliquot into 10 mL of Rappaport Vassiliadis Salmonella
Enrichment Broth, mix, and incubate at 30° to 35° for 18 to 24 hours. Streak a loopful from both incubated media onto individ-
ual surfaces of one or more of following media: Brilliant Green Agar Medium (BG-Agar), Xylose–Lysine–Desoxycholate–Agar Medi-
um (XLDC-Agar), and Hektoen Enteric Agar Medium (HE Agar). Cover, invert the plates, and incubate at 30° to 35° for 24 to 48
hours. Examine the inoculated plates of BG-Agar, XLDC-Agar, and/or HE Agar, and interpret the results with reference to Table
2: if no colonies having the characteristics described are observed, the test specimen meets the requirement for the absence of
Salmonella species. If colonies with characteristics described in Table 2 are present, the suspect colonies are transferred to a
slant of Triple Sugar–Iron–Agar Medium (TSI) using an inoculating wire, by first streaking the surface of the slant, and then stab-
bing the wire well beneath the surface. Incubate at 30° to 35° for 24 to 48 hours. If the tubes do not have red alkaline slants
and yellow acid butts, with or without concomitant blackening of the butts from hydrogen sulfide production, the test speci-
men meets the requirement for the absence of Salmonella species.
Table 2. Characteristics of Salmonella Species on Specified Agar Media
Agar Medium Colonial Morphology Gram Stain
Small, transparent and colorless; or opaque, pink or white
Brilliant Green (often surrounded by pink to red zone) (–), rods
Xylose–Lysine–Desoxycholate Red, with or without black centers (–), rods
Hektoen Enteric Blue-green, with or without black centers (–), rods
Incubate at 30° to 35° for 24 to 48 hours. From FSCD, pipet a 1-mL aliquot into a container containing 10 mL of MacConkey
Broth, mix, and incubate at 42° to 44° for 24 to 48 hours. Streak a loopful from both incubated media onto individual surfaces
of MacConkey Agar Medium (MC Agar), and incubate at 30° to 35° for 18 to 24 hours. Examine the inoculated MC Agar plate,
and interpret the results with reference to Table 3: if no colonies having the characteristics described are observed, the test
specimen meets the requirement for the absence of Escherichia coli. Suspect colonies showing the characteristics described in
Table 3 are transferred individually, using an inoculating loop, to the surface of a plate with Levine Eosin–Methylene Blue–Agar
Medium (LEMB-Agar). If a large number of suspect colonies are to be transferred, divide the surface of each plate into quad-
rants, each quadrant being inoculated with a different colony. Cover the plates, invert, and incubate at 30° to 35° for 24 to 48
hours. If none of the colonies exhibit a characteristic metallic sheen under reflected light, and if none exhibit a blue-black ap-
pearance under transmitted light, the test specimen meets the requirement for the absence of Escherichia coli.
Test Preparation—Prepare as directed for Sampling. [NOTE—On the basis of results for Preparatory Testing, modify the Test
Preparation as appropriate.]
Procedure—Take two equal portions of the Test Preparation, heat one to 80° for 10 minutes, and cool rapidly. Transfer 10
mL of each portion to separate containers, each containing 100 mL of Reinforced Medium for Clostridia, and incubate under
anaerobic conditions at 35° to 37° for 48 hours. After incubation, subculture each specimen on Columbia Agar Medium to
which gentamicin has been added, and incubate under anaerobic conditions at 35° to 37° for 48 hours. Examine the plates,
and interpret with reference to Table 4: if no growth of microorganisms is detected, the test specimen meets the requirement
for the absence of Clostridium species.
Table 4. Characteristics of Clostridium Species on Specified Media
Medium Gram Stain Catalase
Reinforced Medium for Clostridia (+), rods
Columbia Agar (+), rods Negative
If growth occurs, subculture each distinct colony on Columbia Agar Medium, and separately incubate in aerobic and in anae-
robic conditions at 35° to 37° for 48 hours. The occurrence of only anaerobic growth of gram-positive bacilli, giving a negative
catalase reaction, indicates the presence of Clostridium sporogenes. To perform the catalase test, transfer discrete colonies to
glass slides, and apply a drop of dilute hydrogen peroxide solution: the reaction is negative if no gas bubbles evolve. If the test
specimen exhibits none of these characteristics, it meets the requirement for the absence of Clostridium species.
Retest
For the purpose of confirming a doubtful result by any of the procedures outlined in the foregoing tests following their ap-
plication to a 10 g specimen, a retest on a 25 g specimen of the nutritional or dietary supplement may be conducted. Proceed
as directed under Procedure, but make allowances for the larger specimen size.
The raw materials, pharmaceutical ingredients, and active ingredients used in the manufacture of nutritional and dietary arti-
cles may range from chemically synthesized vitamins to plant extracts and animal byproducts, and these ingredients are typi-
cally not sterile. Considerable experience has accrued with these highly refined plant- and animal-derived pharmaceutical in-
gredients, such as microcrystalline cellulose, modified starch, lactose, and magnesium stearate, and their microbiological at-
tributes are well established. Botanicals may be microbiologically contaminated at any point during cultivation, harvesting,
processing, packing, and distribution. Major sources of microbial contamination are associated with human or animal feces
used as plant manure; contaminated irrigation water and/or process water; and poor worker hygiene and sanitation practices
during harvesting, sorting, processing, packaging, and transportation. Furthermore, it is essential that microbiological contam-
ination be minimized during the manufacture of nonsterile dietary supplements. To achieve this, Good Manufacturing Practi-
ces are employed and adequate microbiological specifications are established.
Microbiological process control, control of the bioburden of raw materials, and control of the manufacturing process to min-
imize cross-contamination are necessary to guarantee acceptable microbial quality in the final dosage forms. Because nonaqu-
eous or dry dosage forms do not support microbial growth because of low water activity, the microbial quality of such articles
is a function of the microorganisms introduced through ingredients or during processing. In addition to considering the inten-
ded use of the product, the frequency of microbial testing for the finished nonsterile dietary supplement would be a function
of the historical microbial testing database of that product, knowledge of the manufacturing processes, the susceptibility of the
formulation to microbial proliferation, and the demonstrated effectiveness of programs controlling the raw materials.
From a microbiological perspective, the development of the formulation of nutritional or dietary supplements includes an
evaluation of raw materials and their suppliers and the contribution made to the products by each ingredient and the manu-
facturing processes. Characterization of these elements allows the adequacy of the manufacturing process to be demonstrated.
For example, if a product is formulated with an ingredient of botanical or animal origin known to possess a high, variable, or
unpredictable level of microbiological contamination, it is necessary to ensure that the microbiological monitoring identifies
ingredients that have an inappropriate bioburden level and that a premanufacturing process such as drying, extraction, heat
treatment, irradiation, or gaseous sterilization treatment will inactivate or remove any objectionable contaminant possibly
present.
However, the selected treatment technique should not have any adverse effects. The treatment of raw materials by irradia-
tion and ethylene oxide may cause unwanted changes affecting the safety and efficacy of the raw material. For instance, when
treated by ethylene oxide, crude extracts containing alkaloids have shown reduced contents of alkaloids. Dry heat treatment
has been used for inactivation as well, but requires further evaluation because it may adversely affect stability and degradation
of the raw material. With regard to the design of the manufacturing process, appropriate consideration should be given to the
microbiological effect of wet granulation manufacturing processes. Wetting of a dry powder can result in increased levels of
microorganisms if the granulation is stored prior to drying. However, it is recognized that the pressure and temperature associ-
ated with compression of tablets will decrease microbial counts. Antimicrobial activity is also achieved, especially with aqueous
preparations, by the addition of chemicals that have known antimicrobial properties and that are compatible with the formula-
tion.
However, antimicrobial preservation is not a substitute for Good Manufacturing Practices. A process has to be designed to
minimize the microbiological population. Operating procedures, temperatures, and time limits, including holding times, are
established to protect the product from microbiological contamination and growth. All processes have to be validated for their
intended purposes. Moreover, in-process manufacturing and testing controls necessary for microbiological quality should be
identified and implemented.
Facilities—The facilities, including the building and the heating, ventilation, and air-conditioning (HVAC) systems, should
be designed to minimize microbiological contamination. The design of facilities used for the manufacture of supplements and
their operating parameters should be documented, and the documentation should include, when appropriate, HVAC filter
types, space pressure differentials, temperature, and relative humidity and air changes. Dry products processed in a dry envi-
ronment do not possess a high potential for increased microbial levels. However, some control is warranted to minimize micro-
biological and chemical contamination. Potentially problematic areas are those that utilize Purified Water for wet granulation,
batching liquid products, and film-coating tablets, because water encourages microbial growth.
Equipment—Equipment used for the processing of semisolid and dry supplements should be designed to promote sanitary
conditions, to be self-drying, and to be easy to clean. Dryers, ovens, wet granulation equipment, bulk tanks, and equipment
for preparation of coating solutions are periodically evaluated to ensure that cleaning procedures are adequate.
Water—As one of the major components in nutritional and dietary supplement manufacturing processes, water deserves a
special consideration in the microbiological control of these articles. It is a growth medium for a variety of microorganisms that
present a threat to product quality, safety, preservation, and stability. Water may even act as a carrier of objectionable microor-
ganisms. In view of this, water used in manufacturing is Purified Water. For the manufacture of raw materials, process water
that meets specific microbiological objectives and U.S. Environmental Protection Agency National Drinking Water standards or
equivalent European and Japanese standards may be used.
Cleaning and Sanitization—Detailed and specific cleaning and sanitization procedures should be evaluated, developed,
and validated, with special attention given to product contact surfaces. Personnel should possess sufficient knowledge of these
procedures.
SUPPLEMENT COMPONENTS
Raw materials, excipients, and active substances as components of nutritional and dietary supplements can be a primary
source of microbiological contamination. Specifications should be developed and sampling plans and test procedures should
be employed to guarantee the desired microbiological attributes of these materials. The nature and extent of microbiological
testing should be based upon a knowledge of the material's origin, its manufacturing process, use, historical data, and experi-
ence. For instance, materials of animal or botanical origin that are not highly refined might require special, more frequent test-
ing than synthetic products.
Since members of the family Enterobacteriaceae are a major component of the normal epiphytic and endophytic microflora
(e.g., members of genera Klebsiella, Enterobacter, and Erwinia) and have been associated with the seeds, pods, roots, leaves,
and stems of plants of economic importance, coliform or Enterobacteriaceae counts will not be an appropriate general micro-
biological criterion for botanicals. However, when it is considered advantageous, coliform or Enterobacteriaceae counts may be
included in the individual monographs. Typically on new leaves, bacteria predominate in the microflora, while yeast and fila-
mentous fungi succeed bacteria and become dominant late in the growing season. With dried botanicals, the bacterial popula-
tion will tend to change from Gram-negative bacteria to Gram-positive spore formers and fungi. Refinement of botanicals from
chopped or powdered plant material to powdered extracts using alcoholic, alkaline, acid hydro-alcoholic, or aqueous extract-
ing materials will reduce the likelihood of vegetative microorganisms within the botanical material. The classification of botani-
cal materials is contained in Table 1.
Table 1. Definitions of a Range of Botanical Materials
Botanical Preparation Definition
Hand-picked portions of the botanical (e.g., leaves, flowers, roots, tubers, etc.)
that are air dried, chopped, flaked, sectioned, ground, or pulverized to the con-
Chopped or Powdered Botanicals sistency of a powder.
Extracts are solids or semisolid preparations of a botanical that are prepared by
percolation, filtration, and concentration by evaporation of the percolate. The
extracting material may be alcoholic, alkaline, acid hydro-alcoholic, or aqueous
in nature. Typically, an extract is 4–10 times as strong as the original botanical.
Botanical Extracts The extracts may be semisolids or dry powders termed powdered extracts.
Tinctures are solutions of botanical substances in alcohol obtained by extraction
Tinctures of the powdered, flaked, or sectioned botanical.
Infusions are solutions of botanical principles obtained by soaking the powdered
botanical in hot or cold water for a specified time and straining. Typically, infu-
Infusions sions are 5% in strength.
Decoctions are solutions of botanicals prepared by boiling the material in water
Decoctions for at least 15 min and straining. Typically, decoctions are 5% in strength.
A fluidextract is an alcoholic liquid extract made by percolation of a botanical so
Fluidextracts that 1 mL of the fluidextract represents 1 g of the botanical.
Dried botanicals to which boiling water is added immediately prior to consump-
Botanicals to be treated with boiling water before use tion.
MICROBIOLOGICAL TESTING
Microbiological attribute sampling and testing plans vary widely. In some cases, no sampling or testing is necessary; in other
cases, periodic monitoring is warranted; and yet for some articles, each batch requires sampling and testing. The design of the
sampling and testing plans and the kind of attributes examined depend on the application and the type of the product, the
potential for contamination from components and processing, the growth promotion or inhibition properties of the formula-
tion, and the target population for the supplement. For example, a powdered botanical may have highly variable microbiolog-
ical attributes so that an incoming batch would be sampled and composite testing would not be advised, while a highly re-
fined botanical extract may not require routine microbial testing. Similarly, products with a low water activity will not be sus-
ceptible to microbial growth during their shelf life provided they are protected from elevated humidity by their containers.
See the Introduction under Microbial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ. These tests provide
meaningful information regarding the microbiological acceptability of excipients, active substances, and nonsterile supplement
formulations. If the individual monograph does not specify microbial enumeration limits, the guidance provided in this chapter
is used. Acceptable general limits of microbial levels for raw materials, excipients, and botanical products are shown in Table 2;
and those for raw materials, excipients, active ingredients, and other nonsterile finished articles that are nutritional supple-
ments, but do not contain botanicals, are shown in Table 3.
Table 2. Recommended Microbial Limits for Botanical Ingredients and Products
Recommended Microbial Limit Requirements
Material (cfu/g or mL)
Total aerobic microbial count NMT 105
Total combined yeasts and molds count NMT 103
Bile-tolerant Gram-negative bacteria NMT 103
Dried or Powdered Botanicals Absence of Salmonella spp. and E. coli in 10 g
Total aerobic microbial count NMT 104
Total combined yeasts and molds count NMT 103
Powdered Botanical Extracts Absence of Salmonella spp. and E. coli in 10 g
Total aerobic microbial count NMT 104
Tinctures Total combined yeasts and molds count NMT 103
Total aerobic microbial count NMT 104
Fluidextracts Total combined yeasts and molds count NMT 103
Table 2. Recommended Microbial Limits for Botanical Ingredients and Products (Continued)
Recommended Microbial Limit Requirements
Material (cfu/g or mL)
Total aerobic microbial count NMT 102
Infusions/Decoctions Total combined yeasts and molds count NMT 10
Total aerobic microbial count NMT 104
Total combined yeasts and molds count NMT 103
Nutritional Supplements with Botanicals Absence of Salmonella spp. and E. coli in 10 g
Total aerobic microbial count NMT 106
Total combined yeasts and molds count NMT 104
Bile-tolerant Gram-negative bacteria NMT 102
Botanicals to be treated with boiling water before use Absence of E. coli and Salmonella spp. in 10 g
Table 3. Recommended Microbial Limits for Dietary Supplement Ingredients and Products
Recommended Microbial Limit Requirements
Material (cfu/g or mL)
Total aerobic microbial count NMT 103
Other raw materials and Total combined yeasts and molds count NMT 102
dietary supplement ingredients Absence of E. coli in 10 g
Total aerobic microbial count NMT 103
Nutritional supplements with synthetic or highly refined Total combined yeasts and molds count NMT 102
ingredients Absence of E. coli in 10 g
See Introduction under Microbiological Procedures for Absence of Specified Microorganisms—Nutritional and Dietary Supplements
á2022ñ. Absence of one or more species of objectionable microorganisms is required in some individual monographs.
Test for Aflatoxins—Dietary and nutritional articles containing botanical products with a history of mycotoxin contamina-
tion are also typically tested for aflatoxins, especially if the material is obtained from roots or rhizomes. See Articles of Botanical
Origin á561ñ for the details of a test for aflatoxins. Where necessary, this test is included in the individual monograph.
Solid Oral Dosage Forms—Among all dosage forms, solid oral dosage forms present the lowest microbiological risk be-
cause of their method of manufacture, low water activity, and route of administration. When justified, reduced microbiological
testing may be appropriate.
Other Concerns—The presence of some microorganisms in articles can be an indicator of processes that are not under mi-
crobiological control. For example, Purified Water used at some stage of the manufacture of these products might contain a
typical flora of Gram-negative microorganisms. As with pharmaceutical products, inadequate processing of water and poor
maintenance of water systems may result in the contamination of processed formulations by Gram-negative microorganisms.
This general chapter provides information about several aspects of botanical articles not covered in USP standards mono-
graphs. Although the standards in the monographs address the quality issues associated with botanical plant materials, ex-
tracts, and preparations of Pharmacopeial articles, there is a need to develop appropriate information to optimize the pre-har-
vest conditions for appropriate growth and the post-harvest handling to achieve consistent quality with minimum variations in
the composition of chemical constituents.
PROTOCOL CONTENTS
GENERAL GUIDANCES
It is recommended that, at a minimum, growers and others involved in the handling and distribution of botanical products
should become familiar with and follow the WHO Guidelines for Good Agricultural and Collection Practices (GACP) for Medici-
nal Plants (found at http://www.who.int/medicinedocs/collect/edmweb/pdf/s4928e/s4928e.pdf).
Commercial trade in natural products occurs in a global market. Material of domestic origin must be produced in compli-
ance with all federal laws of the United States. Material of foreign origin, imported into the U.S., must be produced and trans-
ported in compliance with the laws of the U.S., the country of origin, and relevant international treaties. These include, but
may not be limited to, the following:
1. The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) is an international agree-
ment between governments. Its aim is to ensure that international trade in specimens of wild animals and plants does not
threaten their survival. Information about CITES is available at http://www.cites.org.
2. The Convention on Biological Diversity (CBD) establishes three main goals: the conservation of biological diversity, the
sustainable use of its components, and the fair and equitable sharing of the benefits from the use of genetic resources.
Each country that has ratified and is a party to the Convention is responsible for implementation by means of national
enabling legislation that can differ from country to country.
3. The Endangered Species Act (ESA) was originally adopted in 1973. The ESA is a law that aims to protect species of fish,
wildlife, and plants believed to be threatened with extinction. The ESA is administered primarily by the U.S. Fish and Wild-
life Service. Full text of the act is available at: http://epw.senate.gov/esa73.pdf.
Provided below is additional information not covered in the compendial specifications: compendial history; sources; collec-
tion and cultivation, including common adulterants; and drying, storing, and shipping. This information is provided to com-
plement the standards for quality control in the monographs for botanical articles.
Compendial History—The focus in this section is on historical compendial use that has strong validity, with only brief refer-
ence to anecdotal use. This is important information because traditional use is one of the elements taken into consideration to
support the safety and the presumptions of benefits of botanical dietary supplements.
Sources—Included here is the point of origin of the botanical; it also encompasses cultivation (defined as agricultural grow-
ing) and wildcrafting (defined as collected in the wild), along with a listing of the primary geographical (native) areas of pro-
duction.
Collection and Cultivation—This section discusses wildcrafting, the conservation of restricted and rare species, and the
trend to cultivation as an ecological alternative; such optimal harvesting and collection practices serve to preserve the integrity
of species and botanical products. It is divided into three subsections:
1. Collection (conservation and ecology)
2. Cultivation Practices
3. Optimal Times for Harvest
Post-Harvest Handling (Optimal Handling and Processing Practices, Drying, Storage, and Shipping)—Important fac-
tors regarding storage of herbal products and how they should be maintained include the following.
1. Light: Protection from light is important for botanical articles. Light accelerates numerous chemical processes that may
lead to degradation or changes in the constituents of the articles.
2. Temperature: Storage temperatures in this Pharmacopeia are defined in the General Notices. Excessive heat may affect the
content of volatile constituents (essential oils) and accelerate degradation processes. However, heat treatments are some-
times useful in the maintenance of the article's quality and can be used in drying, reducing microbial load, and inhibiting
certain enzymes. Heat application during these processes must be carefully controlled to achieve the desired balance be-
tween degradation and quality conservation.
3. Humidity: Moisture in the articles may allow certain enzymes such as glycosidases to become active, hence degrading
constituents. High humidity also increases the danger of microbial proliferation. As a rule, it is advisable to store botanical
articles below 60% relative humidity. Although controlled humidity and temperature warehouses are now required in
many good manufacturing practices for natural products, much of the world still lacks access to these facilities.
4. Degree of Comminution: The degree of comminution plays a role in determining the stability of the botanical articles dur-
ing storage. The increased surface area in fine powders allows oxidation and other degradation processes to occur more
extensively and rapidly than in the case of a whole article. Plants containing tannins, bitter substances, and essential oils
are particularly sensitive to the degree of comminution. In general, dried crude botanicals should be stored in a minimally
processed form.
5. Containers: Appropriate containers are defined in this Pharmacopeia in the General Notices.
Constituents—Where known, the substances mainly responsible for the activity of the product are listed, along with other
compounds contained in the plant.
Black Cohosh Actaea racemosa L. [Cimicifuga racemosa (L.) Nutt.] (Fam. Ranunculaceae)
Optimal Times for Harvest—Rhizomes and roots should be harvested in autumn when the plant is dormant. At that time the
underground portions of the plant have lower moisture content than in other seasons. Fall harvesting also allows plants to pro-
duce mature seeds before being uprooted.
Post-Harvest Handling—
Optimal Handling and Processing Practices—Rhizomes with roots may be processed fresh or dried. They should be thorough-
ly washed directly after harvest and then laid out to dry. Freshly harvested roots should be solid but not woody.
Drying—Rhizomes with roots are cut and air-dried at 35° to 45°. They are fully dried when they are brittle and snap easily
and when no moisture is evident in cross section, either visibly or to the touch.
Storage—Follow general guidelines for storage by packing in airtight containers protected from light, heat, moisture, and
insect infestation.
Adulterants and Contaminants—Other species of Actaea, especially yellow cohosh (A. podocarpa syn. Cimicifuga ameri-
cana), have commonly been mixed with A. racemosa because of similarity in aboveground appearance and common growing
habitat between species. The two species can be distinguished by differences in their freshly harvested underground parts: the
fresh rhizome of A. podocarpa has a distinct yellowish hue, whereas that of A. racemosa is black. The rhizomes of both species
are far more difficult to tell apart when dry because A. podocarpa darkens upon drying. The underground portions of baneber-
ry (Actaea pachypoda and A. rubra) occur as occasional adulterants of black cohosh supplies. Fruiting plants of baneberry may
be distinguished from black cohosh by their fleshy white or red poisonous berries, which contrast with the dry follicles of black
cohosh. No information was available on how to distinguish the underground portions of black cohosh and baneberry from
each other. According to one herb dealer, the roots of baneberry are smaller than those of black cohosh, and therefore are not
often harvested by wildcrafters. In the Pacific Northwest, Actaea elata (syn. Cimicifuga elata) is collected for medicinal use.
Botanical Identification—Zingiber officinale Roscoe. Herbaceous perennial from tuberous rhizome, aromatic because of the
presence of volatile oils.
Stem: Erect, unbranched pseudostem formed by the tight overlap of sheathing leaf bases; 0.9 to 1.5 m tall.
Leaf: Simple, alternate and two-ranked, sessile or petioles short with bases sheathing the stem and a ligule where the leaf
base meets the stem; blade linear to narrowly lanceolate, 15 to 25 cm long, 1.5 to 3 cm wide; margin entire; glabrous to
pubescent.
Inflorescence: Terminal spike, 3.5 to 8 cm long, 1.5 to 2 cm wide, with conspicuous spirally arranged primary bracts; usu-
ally borne on specialized leafless stems.
Flower: Perfect, bilaterally symmetric; calyx tubular with 3 lobes; corolla tube 2 to 2.5 cm long with lanceolate apical
lobes, 1.5 to 2 cm long, 2 to 3.5 mm wide, greenish yellow; stamen 1, anther cream-colored with dark purple, elongated
connective grasping upper part of style; staminodes 4, petaloid, 2 fused into an erect, ovate-oblong lip that is dull purple with
cream mottling; ovary inferior; style 1, slender, exerted beyond connective.
Fruit: Loculicidal capsule; seeds shiny black with a white aril.
Chromosome number: n = 11.
There are several different varieties and forms of ginger. The varying morphological characteristics of these are displayed in
Table 1.
Table 1. Morphological and Key Characteristics of Ginger from Different Areas of Production
Source Form Aroma Color (External)
Poor quality is recog- Uncut surface dark grayish-
Flat surfaces, mostly peeled, starchy nized by its camphor- brown; cut surface brownish-
Africa and fibrous; 9 cm long, 1.5 cm wide aceous aroma black
Australia Citrus-like Buff
Bengal Flat surfaces, scraped Gray-brown
Short stumpy lobes, unscraped,
China mostly sliced Strong, floral to citrus Pale brown
Cream color with numerous
Cochin Lateral surfaces lacking cork Strong, floral to citrus black resin dots
Up to 12 cm long, 1 cm wide;
Jamaica surfaces completely peeled;
(unbleached) starchy and fibrous thin cortex Delicate, citrus-like All surfaces yellow-brown
Externally gray-white to light
Up to 7 cm long, 12 mm wide; flat grayish-brown, often with
surfaces usually completely peeled; white powder from being
Japan starchy and fibrous thick cortex Bergamot-like coated with lime
Malabar
(Cochin and Cork layer completely removed,
Calcutta) mostly treated with chalk Citrus-like Almost white
Table 1. Morphological and Key Characteristics of Ginger from Different Areas of Production (Continued)
Source Form Aroma Color (External)
Smaller in size than other varieties,
Nigeria rather less deeply scraped Delicate Somewhat darker than other varieties
Compendial History— Ginger was official in the United States Pharmacopoeia from the first edition of 1820 through the
fourteenth revision of 1950, often appearing in multiple preparations. It also appeared in all editions of the United States Dis-
pensatory from 1833 through the final edition of 1973, where it was described as “a stimulant and carminative that has been
used for treatment of dispepsia and flatulent colic”.
Constituents—The essential oils and the pungent principles make up some of the major components of the rhizome of
ginger: 4.0% to 10.0% of the rhizome consists of an oleoresin composed of nonvolatile, pungent principles (phenols such as
gingerols and their related dehydration products, shogaols); nonpungent fats and their waxes. The essential oil (1% to 3%)
contains sesquiterpenes and monoterpenes, mainly geranial and nerals. Generally, but not always, sesquiterpenes predominate
(30% to 70%), such as zingiberene, sesquiphellandrene, and beta-bisabolene, which decompose on drying and storage. The
nonvolatile pungent principles include the phenylalkanones, the gingerols, and the phenylalkanonols, shogaols with varying
chain lengths.
Sources and Distribution—
Sources—Ginger is cultivated in most tropical and subtropical countries to greater or lesser degrees. The world production is
estimated to be 100,000 tons. China and India are reported to be the primary areas of production. Approximately 5000 tons
of ginger are imported into the United States. An estimated 80% of this comes from China. In China, Sichuan and Guizhou
provinces reportedly produce the largest quantities and highest quality. It is also produced in Guangdong, Hubei, Shandong,
Shanxi, and Zhejian provinces. Most of the dried ginger from China available in the United States has had the cortex scraped
or rubbed off before it is dried. This gives it a whitish appearance. The freshly dug root is soaked overnight in water, scraped
with a knife to remove the outer cortex, and then sun-dried. It has been reported that high arsenic levels in the soil of Changn-
ing County of Hunan Province, China, has negatively affected ginger yields.
In India, ginger is grown on a large scale in the warm, moist regions of Madras and Cochin, and to a lesser extent in Bengal
and the Punjab. Varieties grown in Bengal are reportedly the highest quality material in India. Other areas of production in-
clude Africa (Nigeria and Sierra Leone), Australia, the East Indies, Fiji, Hawaii, and Jamaica. The morphological characteristics of
ginger cultivated in these different areas are outlined in Table 1.
In older literature, Jamaican ginger is reported to be the highest quality and the most aromatic, though supplies are limited.
Distribution—Most tropical and subtropical countries, such as Australia, China (Guangdong, Guizhou, Hubei, Shandong,
Shanxi, Sichuan, and Zhejian provinces), India (Bengal, Cochin, Malabar), the East Indies, Fiji, Jamaica, Japan, Nigeria, and Si-
erra Leone. Hawaii in the United States.
Collection and Cultivation—
Collection (Conservation and Ecology)—When the stems wither and are white, the rhizomes are ready for collection. Usually
ginger is harvested after 6 months of growth at the earliest, and sometimes not until as late as 20 months; or to obtain larger
roots, it is harvested in January or February of the second year of growth. In tropical and subtropical areas, roots are harvested
as early as 4 months of growth, because they tend to become fibrous and tough as they get older. As ginger matures, it be-
comes more fibrous and stronger in flavor. Ginger harvest can be described in three stages:
1. Ginger that has been harvested early is known as green ginger and is traded as fresh ginger. It is succulent and tender,
mellow, and mildly aromatic with a floral or lemony aroma and mild flavor.
2. Ginger harvested a few months later is more fibrous and drier and is collected for drying and may be sold as a full-fla-
vored, pungent dried whole ginger.
3. The last harvest is usually around 9 months and yields the strongest ginger, which is quite dry and also richest in pungent
components. This ginger is dried and then ground into powder.
Cultivation Practices—Ginger is a perennial herb that grows well at subtropical temperatures where the rainfall is at least
1.98 meters per year. The plant is sterile and is grown by vegetative means. Selected pieces of rhizome (“seed pieces” or
“setts”), each bearing a bud, are planted in holes or trenches. Ideally the soil should be well-drained, rich clay loam. The grow-
ing conditions resemble those of potato cultivation. Mulching or manuring is necessary because the plant rapidly exhausts the
soil of nutrients.
Ginger is susceptible to waterlogging and root rot. Preventive methods include using only the cleanest ginger for planting
and washing it with fungicide before planting. A study growing ginger hydroponically yielded up to 125 tons per hectare in 6
to 7 months compared to 35 tons per hectare when grown in soil.
Optimal Times for Harvest: typically in December or January.
Post-Harvest Handling—
Optimal Handling and Processing Practices—After harvesting, the rhizome is cleaned and stripped of its stems and roots. Each
area processes its ginger differently after harvest. This results in the different quality and commercial grades available on the
market. Green ginger consists of the rhizomes sent to market without drying. Unscraped or partially scraped varieties are tra-
ded as coated or black ginger. These roots have been scalded with boiling water and dried quickly. When dry, black ginger
breaks with a horny, blackish, somewhat diaphanous fracture, due to the pasty condition of the starch. White ginger is bleach-
ed, usually by rubbing with chalk or lime, to lighten its color and to prevent insect infestation. Preserved ginger consists of
soft, yellowish-brown pieces obtained by steeping the fresh ginger in hot syrup and carefully bottling. It is soft, brown-yellow
and translucent. When baked, ginger loses its pungency and acquires a bitter taste.
Drying—In general, after harvest, the fresh roots are washed, and the whole dark outer skin, consisting of cork and a little
underlying parenchyma, is scraped away. Scraping speeds up the drying time of the crude drug. However, excessive scraping
can result in lower concentrations of essential oil that is lost with the discarded epidermal tissue. After scraping, the rhizomes
are then laid out on clean floors and dried in the sun for 7 to 10 days. During this time they are occasionally turned and are
piled up every night. If the fresh rhizomes are too fleshy or moist, drying will take longer and the product will end up looking
shriveled. To obtain a whiter product, the ginger is moistened after 5 or 6 days and dried for another 2 days, at which time it
is ready for export. Dried ginger is more pungent and stronger in taste than fresh ginger.
Storage—Store in a tightly closed container, protected from light and moisture, in a cool area. A study was done on ginger
harvested after 8, 9.5, 11, or 12 months. Samples were stored at 10° to 15° and 45% to 55% relative humidity or 25° to 30°
and 75% relative humidity for 0, 4, or 8 weeks. Oil and oleoresin yields increased with the age of the ginger. Room tempera-
ture storage had adverse effects, but refrigerated storage for up to 4 weeks had no effect on quality. When stored for extended
periods of time, ground ginger loses its pungency.
Adulterants—Because ginger is so characteristic, unintentional adulterants are rare. However, in East Asia sometimes the
much larger Zedoary cassumer and Zedoary zerumbet, along with Alpinia allughas, are used and found in European commerce.
They are easy to distinguish because of their characteristic aromas. Occasionally, Chinese sugar-candied “ginger” is prepared
from Alpinia galangal.
In older literature, other herbs have reportedly been used as adulterants. These include various species of Curcuma, Capsi-
cum, and Grains of Paradise (Amomum melegueta) added to exhausted material in order to enhance color and pungency.
Ginger powder is sometimes adulterated with plant starches such as those from wheat middlings, potatoes, corn, barley,
rice, legumes, acorns, flaxseed meal, mannihot, oil cakes from linseed, rapeseed, mustard, almond meal, palm kernel or olives,
hazelnut shells, and mineral additives. These may be easily identified microscopically. The extent of this type of adulteration in
trade is unknown.
Exhausted material should be considered an adulterant.
and sesquiterpene derivatives. The amount of valepotriates present also varies widely between species and genera and even
within a species, generally ranging from 0.5% to 1.2%. Valepotriates are particularly unstable; they decompose easily under
the effect of moisture, temperatures above 40°, or acidity (pH <3).
Valerian also contains small amounts of aliphatic acids, alkaloids, amino acids, phenolic acids, flavonoids, free fatty acids,
sugars, and salts. Valerian constituents that have possible sedative effects include acetoxyvalerenic acid, 1-acevaltrate, baldri-
nal, didrovaltrate, hydroxyvalerenic acid, kessane derivatives, valeranone, valerenal, valerenic acid, and valtrate.
Sources and Distribution—
Sources—Valerian is found in damp or dry meadows, scrub, or woods in most of Europe, although rare in the south, and it
is cultivated and naturalized in North America. Valerian is cultivated in Belgium, Britain, Eastern Europe, France, Germany, Ja-
pan, the Netherlands, North America, and Russia. The majority of standardized extract products and crude cut and sifted ma-
terial on the domestic market are prepared from European supplies. A large number of liquid extracts are prepared from do-
mestically cultivated material. Many species other than V. officinalis are reported to be traded as medicinal valerian. These in-
clude V. edulis Nutt. ex Torr. & A. Gray, V. corneana Briq. k, V. stubendorfi Kreyer ex Kom., V. amurensis P. Smirn. ex Kom., V.
hardwickii Wall., V. exaltata Mikan, and V. wallichi DC. syn. V. jatamansi Jones.* The most frequently used North American spe-
cies include V. sitchensis Bong and V. edulis Nutt.* = V. edulis Nutt. ex Torr. & Gray ssp. procera. Other species reported to be
used locally include V. arizonica Gray, V. capitata Pall ex Link., V. diocia L., and V. scouleri Rydb. Detailed chemical analyses of
most American species are lacking. A limited number of assays of material cultivated in the Pacific Northwest show varying
levels of essential oil ranging from 0.4% to 1.3%. Valerenic acid and valepotriates have been found to be present in fresh and
dry samples of V. sitchensis Bong. V. sitchensis Bong exhibits a strong pungency when fresh. High quality material is reported to
contain from 1.0% to 1.5% essential oil, ³30% extractable matter, and ³0.5% valerenic acid.
Distribution—Europe (Belgium, Britain, Eastern Europe, France, Germany, the Netherlands), Japan, North America, and Rus-
sia.
Collection and Cultivation—
Collection (Conservation and Ecology)—The majority of valerian in trade comes from cultivated material. Harvest times will
vary geographically. The composition of the essential oil varies greatly among different populations of the same subspecies
and even between the same population of plants from year to year. Essential oil content also varies with genotypes, harvest
times, growing conditions, age of root, drying techniques, and method of analysis. It has been reported that valerian harves-
ted in higher elevations, grown in dryer regions, or cultivated in phosphate-rich soil yields relatively high levels of essential oil.
Older literature reports that valerian should be harvested in the fall, between August and September, preferably in the sec-
ond year of growth. Analyses of material cultivated in the Netherlands report that the majority of constituents, including the
essential oil and valerenic acid, were highest in roots harvested in the first year of growth, with essential oil being highest in
September and November (1.2% to 2.1%). The next highest level of essential oil was reported for material harvested in March
(0.9% to 1.6%). Valerenic acid and its derivatives were found to be highest in February and March (0.7% to 0.9%), followed
by material harvested in September (0.5% to 0.7%) and then in January (0.3% to 0.4%). From a commercial standpoint, it is
more cost effective to harvest the roots in the same year the plants are sown than in the second year.
Cultivation Practices—Sowing seeds has been reported to be preferred over planting of seedlings. Best results were achieved
by flat field planting at row spacings of 50 cm and a seed rate of 3 kg per hectare. Cutting off the flowering tops before the
plant has set seed causes the rhizome to develop more fully.
Optimal Times for Harvest—Wagner reports that harvest should take place in the morning during relatively cool weather, a
general recommendation for roots rich in essential oils.
Post-Harvest Handling—
Optimal Handling and Processing Practices—The essential oil is located in the hypodermis of the rhizome in large thin-walled
cells. Therefore, care must be taken not to damage these cells during handling. Excess washing of the roots can result in a
significant reduction of extractive matter. Because of the sensitivity of volatile oils to heat, it is necessary to minimize the
amount of time generated in the grinding or powdering process by doing small lots at a time, with frequent interruptions in
run times, or by using a cryogenic grinder.
Drying—For maximum preservation of the essential oils, valerian should be dried at 40° with a flow rate of 0.05 kg per sec
per m2. Alternatively, drying at 20° for approximately 10 days, shade drying at approximately 45°, low temperature vacuum-
drying, and freeze-drying are also reported to be appropriate drying techniques.
Careless or prolonged drying produces a darker color in the roots and results in the hydrolysis of the isovalerianic esters and
the liberation of isovaleric and hydroxyisovaleric acid. This produces the characteristic valerianic aroma. Properly dried valerian
will produce this same aroma over time.
Storage—Store in closed containers protected from light, air, and moisture. Hydroxyvalerenic acid, a decomposition prod-
uct of acetoxyvalerenic acid, is formed when the herb is stored at too high humidity.
Improper storage conditions can cause significant deterioration of the material. Although the essential oil is relatively stable,
it can evaporate with excessive exposure to air. The essential oil can degrade quickly in powdered material. In powdered root,
the essential oil content can decrease by 50% within 6 months.
Valepotriates are sensitive to humidity, temperatures above 40°, and acid media (pH <3) and are generally not detected in
commercial products after 60 days.
* V. wallichi DC. and V. edulis Nutt. reportedly are lacking in valerenic acid and its derivatives.
Official text. Reprinted from First Supplement to USP38-NF33.
810 á2030ñ Supplemental Information / Dietary Supplements DSC
Adulterants—Other species of valerian: An unidentified Apiaceae species may be found in valerian trade. Adulteration of
valerian in the American market is not common. Many species other than V. officinalis are reported to be traded as medicinal
valerian. These include V. edulis Nutt. ex Torr. & A. Gray, V. coreana Briq. k, V. stubendorfi Kreyer ex Kom., V. amurensis P.
Smirn. ex Kom., V. hardwickii Wall, V. exaltata Mikan, and V. wallichi DC. syn. V. jatamansi Jones.
Botanical Identification—Ulmus rubra Muhlenberg; tree to 35 m high, with spreading branches and open flat crown;
preparations derived from inner bark. U. rubra appears to be more closely related to the introduced Asian species U. pumila L.
than to other native American species of Ulmus; where the two co-occur, interbreeding is common.
Trunk: 18 to 35 m high, to 1 m in diameter; the trunk rises free of branches until about 5 to 6 m.
Branches: Erect, spreading; young twigs are scabrous-pubescent.
Bark: Dark brown to reddish-brown, deeply furrowed. Inner bark is whitish (outer surface yellow-orange; inner surface
pale yellow), fragrant (upon powdering, a distinctive fenugreek-like odor) and very mucilaginous upon chewing or moistening.
Leaves: Alternate; simple; petiolate with petiole (3–)5–7(–9) mm long; 7–18(–23) cm long, 5–10(–15) cm broad; elliptical
to ovate, oblong, or obovate with oblique base and acuminate apex; margins serrate toward base, elsewhere doubly serrate;
upper surface scabrous, rough; lower surface tomentose; secondary veins parallel, slightly curved, running to tips of marginal
teeth.
Inflorescence: Axillary fascicles, roughly hemispherical, to 1.5(–2.5) cm in diameter.
Flowers: Small, perfect; pedicels 1–2(–3) mm long; calyx campanulate, 5–9-lobed at apex, about 2.6–3.5 mm in diameter,
reddish-pubescent; petals absent; stamens 5–9, exserted at flowering; styles 2. Flowers occur before the leaves from March
through early May.
Fruit: Winged samara, yellowish, irregularly suborbicular or occasionally broadly elliptical or obovate, 10–20 mm in diame-
ter, reddish-pubescent over seed; wing papery-textured.
Compendial History—Slippery elm (Ulmus) inner bark appeared in the list of materia medica in the first United States Phar-
macopoeia (USP) of 1820 and remained official until it was removed from USP XI (1936). The USP 1820 included instructions
for the preparation of Infusion of Slippery Elm: “Take of Slippery elm, sliced, one ounce. Boiling water, one pint. Infuse for twelve
hours in a covered vessel, near the fire with frequent agitation, and strain.” Immediately following its removal from USP XI (offi-
cial: June 1, 1936), Slippery Elm Bark became an official monograph in the sixth edition of the National Formulary (NF; official:
June 1, 1936) until its elimination from the 11th edition (official: October 1, 1961). It became official again, as Elm, on Novem-
ber 15, 1995, in the USP section of the Third Supplement to the United States Pharmacopeia–National Formulary (USP 23–NF 18).
A revision was published in the Seventh Supplement on November 15, 1997.
In 1982, Elm Bark appeared in the Food and Drug Administration (FDA) Advance Notice of Proposed Rulemaking (ANPR) for
the establishment of a therapeutic monograph for oral health care drug products for over-the-counter (OTC) human use. In
the ANPR (1982) as well as in the subsequent tentative final monograph of 1988 and in the amendment to the monograph of
1991, Elm bark was classified as a Category I (Generally Recognized as Safe and Effective (GRASE)) OTC oral demulcent active
ingredient, and appropriate standards were urged to be developed in the official compendia.
Aside from USP–NF, the monograph of Elm had already appeared in the second edition of The Dispensatory of the United
States of America (1834), and its last appearance was in the 25th edition, 1960.
Constituents—Constituents of relevance for conformance to Identification A under Elm are mucilaginous substances. Elm
inner bark mucilage is readily extractable by water and consists principally of a polysaccharide which on hydrolysis yields D-
galactose, D-methyl galactose, L-rhamnose, and glucose. Borohydride reduction of the periodate-oxidized polysaccharide af-
fords, on partial hydrolysis with hot acid, three oligosaccharides: O-(3-O-methyl-b -D-galactopyranosyl)-(1®4)-O-(3-O-methyl-
b -D-galactopyranosyl)-(1®4)-L-rhamnose, O-(3-O-methyl-b -D-galactopyranosyl)-(1®4)-L-rhamnose, and O-(3-O-methyl-b -D-
galactopyranosyl)-(1®4)-3-O-methyl-D-galactose.
Other Elm constituents are traces of tannins, including proanthocyanidins, some starch, traces of oxalate salts, beta-sitoster-
ol, and minerals.
Sources and Distribution—
Sources—Slippery elm bark is harvested from wild populations in eastern Canada and the United States, from southern Que-
bec west to North Dakota, south to south-central Texas, and Florida. It is common throughout eastern, southern, and mid-
western U.S., and it grows in more than 25 states. An increasing amount of the commercial supply is being collected accord-
ing to sustainable wild resource management plans as a condition of organic certification for wild crops. Conversely, Dutch
elm disease has had a significant negative impact on elm populations, from 1930 when it was first found in the United States
affecting over 50% of elm trees in the northern states.
Distribution—Canada (New Brunswick, Ontario, Quebec), the United States (Alabama, Arkansas, Connecticut, Delaware, the
District of Columbia, Florida, Georgia, Illinois, Indiana, Iowa, Kansas, Kentucky, Louisiana, Maine, Maryland, Massachusetts,
Michigan, Minnesota, Mississippi, Missouri, New Hampshire, New Jersey, New York, North Carolina, North Dakota, Ohio,
Oklahoma, Pennsylvania, Rhode Island, South Carolina, South Dakota, Tennessee, Texas, Virginia, Vermont, Wisconsin, and
West Virginia).
quality (e.g., NMT 2% of adhering outer bark, NMT 2% foreign organic matter, NMT 10% total ash, and NMT 0.65% acid-
insoluble ash), a clean bark rosser (hand tool with handle and knife blade) should be used to shave off the outer bark. The
rough, scaly matter on the surface of the bark is called ross, and to ross bark is to scrape or shave the outer bark from the limb.
An experienced rosser can visually discern that at least 98% of the outer bark has been shaved off. The inner bark is white in
color (in the spring; reddish later in the season) in obvious visible contrast to the brown outer bark layer. After most of the
outer bark is rossed off, greater care must be exercised to very carefully slice off the remaining thin layer of outer bark so as not
to waste any of the inner bark in the process. After removal of the outer bark, the inner bark can then be removed in strips,
squares, or chips. An incision is made with a clean knife down the center of the limb. Then a clean crowbar is slipped under-
neath the incision in order to lift and peel the inner bark off from the cambium. The strips of inner bark are stacked on a clean
tarp and later bundled for transport to the drying facility.
Drying—Elm USP requires a loss on drying limit of NMT 12%. So long as rain is not expected, fresh elm inner bark can be
sun-cured within a temperature range of 32° to 60°. Drying can also be carried out in a warm room with airflow or in a green-
house. Greenhouse drying takes about 3 to 4 days. Drying indoors can take 5 to 7 days, depending on the heat source. Drying
at commercial scale, however, is done typically in enclosed drying chambers, in which time and temperature can be better
controlled. The strips of elm bark are placed onto a clean screen floor and dried over about 2 days’ time at about 50° with fan-
forced heat through the floor. Because of additional phytosanitary requirements for export of tree barks to Europe, higher heat
exposure is necessary, usually at least 65° but up to 93° for up to two days. Post-drying, the strips of inner bark can be cut or
sawn into pieces of equal length and bound into bundles with wire. The bundles usually consist of flat, oblong pieces, about
30 cm in length and from 10 to 15 cm in width. The bark strips can be stored this way until further processing (e.g., cutting or
powdering) is scheduled.
Storage—To maintain pharmacopeial purity and quality (e.g., to prevent accumulation of excess moisture), dried elm inner
bark should be preserved in well-closed containers, and stored in a cool, dry place.
Adulterants and Contaminants—Common contaminants that could cause a material not to conform with the identifica-
tion tests in the Elm monograph in USP would include other plant parts: for example, greater than 2% outer bark, which lacks
mucilage. Insufficient shaving or rossing of outer bark could cause the material to exceed the monograph limit of NMT 2% of
adhering outer bark. Other possible contaminants would include visible discolored inner bark, although no maximum limit has
been established (for example, inner bark with visible black streaking obtained from a diseased tree). Powdered bark can also
be adulterated with cornmeal, rice flour, starch, or other starchy substances. Consequences of contamination with outer bark
or adulteration with flour or starch are lower mucilage content, lower swelling index value, and correspondingly less of a ther-
apeutic demulcent effect that is mucilage-dependent. Excess outer bark could also cause the material to fail the quantitative
standard of NMT 10% total ash. Methods to determine the presence of adulterants include microscopic examination in order
to determine the presence of excess outer bark or any other adulterant and the concentration of mucilage cells. The Elm muci-
lage test (Identification A) as well as a modified swelling volume test (based on the test in the USP monograph Psyllium Husk)
may also be useful to investigate if adulteration is suspected.
INTRODUCTION
This general chapter is provided to determine compliance with the disintegration and dissolution standards for dietary sup-
plements where stated in the individual monographs.
For the purposes of this chapter, dietary supplement dosage forms have been divided into three categories: Vitamin–Mineral
Dosage Forms, Botanical Dosage Forms, and Dietary Supplements Other Than Vitamin–Mineral and Botanical Dosage Forms. Vita-
min–Mineral Dosage Forms includes articles prepared with vitamins, minerals, or combinations of these dietary ingredients, as
described in Table 1. Botanical Dosage Forms comprises formulations containing ingredients of botanical origin, including plant
materials and extracts. Dietary Supplements Other Than Vitamin–Mineral and Botanical Dosage Forms encompasses dietary sup-
plements formulated with lawfully recognized dietary ingredients that are different from those pertaining to the two foregoing
categories (e.g., amino acids, chondroitin, and glucosamine.)
Where a dietary supplement represents a combination of the categories mentioned above, and there is a difference between
the requirements for the individual categories, the more stringent requirement applies. [NOTE—“More stringent requirement”
means stricter acceptance criteria and/or milder operational conditions.]
Disintegration and dissolution tests as described in this chapter are quality-control tools to assess performance characteristics
of dietary supplement finished dosage forms. These performance standards are intended to detect problems that may arise
due to use or misuse, or changes in coatings, lubricants, disintegrants, and other components. These performance tests are
also intended to detect manufacturing process issues such as over-compression and over-drying that would affect the release
characteristics of the final dosage forms. These tests are not intended to be used as a demonstration or as a surrogate for in
vivo absorption, bioavailability, or effectiveness, unless an in vitro–in vivo correlation (IVIVC) has been established.
DISINTEGRATION
This test is provided to determine whether dietary supplement tablets or capsules disintegrate within the prescribed time
when placed in a liquid medium at the experimental conditions presented below. Compliance with the limits on Disintegration
stated in the individual monographs for dietary supplements is required except where the label states that the products are
intended for use as troches, are to be chewed, or are designed as extended-release dosage forms. Dietary supplements claim-
ing to be extended-release dosage forms must comply with standards other than disintegration to verify that the release of the
dietary ingredients from the dosage form is for a defined period of time. Dietary supplements claiming to be extended-release
dosage forms shall not be labeled as in compliance with USP unless a USP monograph exists for such product. Determine the
type of units under test from the labeling and from observation, and apply the appropriate procedure to 6 or more units.
For purposes of this test, disintegration does not imply complete solution of the unit or even of its active constituent. Com-
plete disintegration is defined as that state in which any residue of the unit, except fragments of insoluble coating or capsule
shell, remaining on the screen of the test apparatus or adhering to the lower surface of the disk, if used, is a soft mass having
no palpably firm core.
Apparatus
Apparatus A: Use the Apparatus described under Disintegration á701ñ for tablets or capsules that are NMT 18 mm long.
For larger tablets or capsules, use Apparatus B.
Apparatus B: The apparatus1 consists of a basket-rack assembly, a 1000-mL low-form beaker for the immersion fluid, a
thermostatic arrangement for heating the fluid between 35° and 39°, and a device for raising and lowering the basket in the
immersion fluid at a constant frequency rate between 29 and 32 cycles per min through a distance of 53–57 mm. The volume
of the fluid in the vessel is such that at the highest point of the upward stroke the wire mesh remains at least 15 mm below the
surface of the fluid and descends to NLT 25 mm from the bottom of the vessel on the downward stroke. At no time should the
top of the basket-rack assembly become submerged. The time required for the upward stroke is equal to the time required for
the downward stroke, and the change in stroke direction is a smooth transition rather than an abrupt reversal of motion. The
basket-rack assembly moves vertically along its axis. There is no appreciable horizontal motion or movement of the axis from
the vertical.
Basket-rack assembly: The basket-rack assembly (see Figure 1) consists of three open-ended transparent tubes, each 77.5 ±
2.5 mm long and having an inside diameter of 32.0–34.6 mm and a wall 2.0–3.0 mm thick; the tubes are held in a vertical
position by two plastic plates, each 97 ± 2 mm in diameter and 7.5–10.5 mm in thickness, with three holes, 36.0–40.6 mm in
diameter, equidistant from the center of the plate and equally spaced from one another. Attached to the undersurface of the
lower plate is 10-mesh No. 23 (0.025-inch) W. and M. gauge woven stainless-steel wire cloth having a plain square weave.
The parts of the apparatus are assembled and rigidly held by means of three bolts passing through the two plastic plates. A
suitable means is provided to suspend the basket-rack assembly from the raising and lowering device, using a point on its axis.
The design of the basket-rack assembly may be varied somewhat, provided that the specifications for the glass tubes and the
screen mesh size are maintained.
Beaker: Low form, 1000 mL; the difference between the diameter of the plastic plates, which hold the tubes in a vertical
position, and the inside diameter of the beaker should be NMT 6 mm.2
Disks: Each tube is provided with a perforated cylindrical disk 15.3 ± 0.15 mm thick and 31.4 ± 0.13 mm in diameter. The
disk is made of a suitable, transparent plastic material having a specific gravity of between 1.18 and 1.20. Seven holes 3.15 ±
0.1-mm in diameter extend between the ends of the cylinder, one of the holes being in the center and the other six parallel
with it and spaced equally tangent to a circle with a radius of 4.2-mm from the center of the disk. All surfaces of the disk are
smooth.3
1 An apparatus and disks meeting these specifications are available from Varian Inc., 13000 Weston Parkway, Cary, NC 27513, or from laboratory supply houses.
2 1000-mL low-form beakers, designed in compliance with the current ASTM E 960 Type I or Type II or ISO 3819 specifications, meet the size requirements.
3 The use of automatic detection using modified disks is permitted where the use of disks is specified or allowed. Such disks must comply with the requirements
Procedure
Test 6 dosage forms as described below for each dosage type. [NOTE—Two basket arrangements for a total of 6 tubes are
necessary for Apparatus B.] If 1 or 2 dosage forms fail to disintegrate completely, repeat the test on 12 additional dosage
forms.
Uncoated tablets: Place 1 tablet in each of the tubes of the basket and, if prescribed, add a disk to each tube. Operate
the apparatus, using water or the specified medium as the immersion fluid, maintained at 37 ± 2°. At the end of 30 min, lift
the basket from the fluid, and observe the tablets.
Plain coated tablets: Place 1 tablet in each of the tubes of the basket and, if the tablet has a soluble external sugar coat-
ing, immerse the basket in water at room temperature for 5 min. Then, if prescribed, add a disk to each tube, and operate the
apparatus, using water or the specified medium as the immersion fluid, maintained at 37 ± 2°. At the end of 30 min, lift the
basket from the fluid, and observe the tablets.
Delayed-release (enteric-coated) tablets: Omit the use of a disk. Place 1 tablet in each of the 6 tubes of the basket, and
if the tablet has a soluble external sugar coating, immerse the basket in water at room temperature for 5 min. Then operate
the apparatus using simulated gastric fluid TS maintained at 37 ± 2° as the immersion fluid. After 1 h of operation in simulated
gastric fluid TS, lift the basket from the fluid, and observe the tablets: the tablets show no evidence of disintegration, cracking,
or softening. Operate the apparatus, using simulated intestinal fluid TS, maintained at 37 ± 2°, as the immersion fluid for the
time specified in the monograph. Lift the basket from the fluid, and observe the tablets.
Delayed-release (enteric-coated) soft shell capsules: Place 1 softgel capsule in each of the 6 tubes of the basket. Omit
the use of a disk. Operate the apparatus using simulated gastric fluid TS maintained at 37 ± 2° as the immersion fluid. After 1 h
of operation in simulated gastric fluid TS, lift the basket from the fluid and observe the softgels: the softgels show no evidence
of disintegration or rupture permitting the escape of the contents. Operate the apparatus with disks, using simulated intestinal
fluid TS, maintained at 37 ± 2°, as the immersion fluid for NMT 60 min. Lift the basket from the fluid, and observe the capsu-
les.
Hard shell capsules: Apply the test for Uncoated tablets, using as the immersion fluid, maintained at 37 ± 2°, a 0.05 M
acetate buffer prepared by mixing 2.99 g of sodium acetate trihydrate and 1.66 mL of glacial acetic acid with water to obtain
a 1000-mL solution with a pH of 4.50 ± 0.05. Attach a removable wire cloth, as described in Basket-rack assembly, to the sur-
face of the upper plate of the basket-rack assembly. At the end of 30 min, lift the basket from the fluid, and observe the capsu-
les.
Soft shell capsules: Proceed as directed in Rupture Test for Soft Shell Capsules.
Use of Disks
Vitamin–mineral dosage forms: Add a disk to each tube unless otherwise specified in the Procedure above or in the indi-
vidual monograph.
Botanical dosage forms: Omit the use of disks unless otherwise specified in the Procedure above or in the individual mon-
ograph.
Dietary supplements other than vitamin–mineral and botanical dosage forms: Omit the use of disks unless otherwise
specified above or in the individual monograph.
Tolerances
All of the 6 dosage forms initially tested or NLT 16 of a total of 18 dosage forms tested disintegrate completely.
DISSOLUTION
This test is provided to determine compliance with the Dissolution requirements where stated in the individual monograph
for dietary supplements, except where the label states that tablets are to be chewed. The operative assumption inherent in this
test is that if the index vitamin or mineral or marker compound(s) for a botanical is dissolved within the time frame and under
conditions specified, the dosage form does not suffer from formulation or manufacturing-related problems affecting the ade-
quate release of the active ingredients.
Apparatus
See á711ñ for description of apparatus used, Apparatus Suitability Test, and other related information.
Figure 2 shows the schematic view of a flow-through cell (USP Apparatus 4) specifically intended for dissolution of lipid-filled
soft shell capsules. The lower part (1) is made up of two adjacent chambers connected to an overflow device. The dissolution
medium passes through chamber A and is subjected to an upward flow. The flow in chamber B is directed downward to a
small-size bore exit that leads upward to a filter assembly. The middle part (2) of the cell has a cavity designed to collect lipo-
philic excipients that float on the dissolution medium. A metal grid serves as a rough filter. The upper part (3) holds a filter
unit for paper, glass fiber, or cellulose filters.
Figure 2. Flow-through cell designed for lipid-filled soft gelatin capsules (dimensions in mm).
Of the types of apparatus described in á711ñ, use the one specified in the individual monograph.
For hard or soft gelatin capsules and gelatin-coated tablets that do not conform to the dissolution specification, repeat the
test as follows. Where water or a medium with a pH of less than 6.8 is specified as the Medium in the individual monograph,
the same Medium specified may be used with the addition of purified pepsin that results in an activity of 750,000 Units or less
per 1000 mL. For media with a pH of 6.8 or greater, pancreatin can be added to produce NMT 1750 USP Units of protease
activity per 1000 mL.
All dietary supplement tablets or capsules containing folic acid are subject to the dissolution test and criteria for folic acid
described in this chapter. This test is required because of the importance of the relationship between folate deficiency and the
risk of neural tube defects. Dietary supplements tablets or capsules containing water-soluble vitamins, minerals, or their combi-
nation are subject to the dissolution test and criteria for index vitamins, index minerals, or both described in this chapter. Diet-
ary supplement tablet and hard shell capsule with solid contents dosage forms containing vitamin A are subject to the dissolu-
tion test and criteria for vitamin A described in this chapter. Dissolution standards were not established and therefore not ap-
plicable to vitamin A in dietary supplement soft shell capsules filled with liquids. Table 1 summarizes the dissolution require-
ments for the assigned USP classes of dietary supplements. Vitamin–mineral combinations that don't belong to any of the USP
classes listed in Table 1 are subject to the Dissolution test and criteria specified in the individual monographs.
Table 1. Dietary Supplements—Vitamin–Mineral Dosage Forms
Dissolution
Requirements Dissolution
for Tablets and Hard Shell Requirements
USP Capsules for Soft Shell Capsules
Class Ingredients with Solid Contents Filled with Liquids
Oil-Soluble
I Vitamins Vitamin A (if present) Not applicable
Water-Soluble One index water-soluble vitamin and folic One index water-soluble vitamin and folic
II Vitamins acid (if present) acid (if present)
Water-Soluble One index water-soluble vitamin, one One index water-soluble vitamin, one index
III Vitamins with Minerals index element, and folic acid (if present) element, and folic acid (if present)
Vitamin A (if present), one index water- One index water-soluble vitamin and folic
IV Oil- and Water-Soluble Vitamins soluble vitamin, and folic acid (if present) acid (if present)
Vitamin A (if present), one index water-
Oil- and Water-Soluble Vitamins with soluble vitamin, one index One index water-soluble vitamin, one index
V Minerals element, and folic acid (if present) element, and folic acid (if present)
Compliance with the dissolution requirements for dietary supplements representing combinations of water-soluble vitamins
(Water-Soluble Vitamins Capsules and Water-Soluble Vitamins Tablets) and combinations of oil- and water-soluble vitamins (Oil-
and Water-Soluble Vitamins Capsules and Oil- and Water-Soluble Vitamins Tablets) is determined by measuring the dissolution of
a single index vitamin from the water-soluble vitamins present. Riboflavin is the index vitamin when present in the formula-
tion. For formulations that do not contain riboflavin, pyridoxine is the index vitamin. If neither riboflavin nor pyridoxine is
present in the formulation, the index vitamin is niacinamide (or niacin), and in the absence of niacinamide (or niacin), the
index vitamin is thiamine. If none of these four water-soluble vitamins is present in the formulation, the index vitamin is ascor-
bic acid.
Compliance with the dissolution requirements for dietary supplements representing combinations of minerals (Minerals Cap-
sules and Minerals Tablets) is determined by measuring the dissolution of only one index element. Iron is the index element
when present in the formulation. For formulations that do not contain iron, the index element is calcium. If neither iron nor
calcium is present, the index element is zinc. In the absence of all three of these elements, magnesium is the index element.
Compliance with the dissolution requirements for dietary supplements representing combinations of water-soluble vitamins
and minerals (Water-Soluble Vitamins with Minerals Capsules and Water-Soluble Vitamins with Minerals Tablets) and combinations
of oil- and water-soluble vitamins and minerals (Oil- and Water-Soluble Vitamins with Minerals Capsules and Oil- and Water-Solu-
ble Vitamins with Minerals Tablets) is determined by measuring the dissolution of one index water-soluble vitamin and one in-
dex element, designated according to the respective hierarchies described above.
Test 1
Medium: 0.1 N hydrochloric acid; 900 mL
PROCEDURES
In the following procedures, combine equal volumes of the filtered solutions of the 6 individual specimens withdrawn, and
determine the amount of vitamin A, folic acid, or the index vitamin or element dissolved, based on the average of 6 units tes-
ted. Make any necessary modifications including concentration of the analyte in the volume of Sample solution taken. Use the
Medium for preparation of the Standard solution and dilution, if necessary, of the Sample solution.
Vitamin A: Determine the percentage of retinyl acetate or retinyl palmitate dissolved by using the following procedure.
Sample solution: Withdraw a portion of the solution under test, pass through a suitable filter of 0.45-mm pore size, and
use the pooled sample as the test specimen.
Standard solution: Dissolve a suitable amount of USP Retinyl Acetate RS or USP Retinyl Palmitate RS in isopropyl alcohol,
and dilute with Medium to obtain a concentration similar to that expected in the Sample solution. [NOTE—The amount of alco-
hol should be 5%–10%.]
Solution A: Methanol and water (90:10)
Solution B: Methanol and isopropyl alcohol (55:45)
Mobile phase: See Table 2.
Table 2
Time Solution A Solution B
(min) (%) (%)
0 100 0
8 0 100
13 0 100
13.1 100 0
15 100 0
Chromatographic system
(See Chromatography á621ñ, System Suitability.)
Mode: LC
Detector: UV 325 nm
Column: 4.6-mm × 10-cm; 3-mm packing L1
Flow rate: 1.0 mL/min
Injection volume: 50 mL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 1.5 for retinyl acetate and NMT 2.0 for retinyl palmitate
Relative standard deviation: NMT 2.0%
Analysis
Samples: Appropriate Standard solution and Sample solution
Result = (rU/rS) × [CS × (V/L)] × 100
TOLERANCES
The requirements are met if NLT 75% of the labeled content of vitamin A, NLT 75% of the labeled content of folic acid, and
NLT 75% of the labeled content of the index vitamin or the index element from the units tested is dissolved.
Compliance with dissolution requirements necessitates the testing of 6 dosage units individually, or testing 2 or more dos-
age units in each of the 6 vessels of the dissolution apparatus, and measuring the dissolution of 1 or more index/marker com-
pound(s) or the extract specified in the individual monograph.
PROCEDURES
Combine equal volumes of the filtered solutions of the 6 or more individual specimens withdrawn, and use the pooled sam-
ple as the Sample solution. Determine the average amount of index or marker compound(s) or the extract dissolved in the
pooled sample by the procedure specified in the individual monograph. Make any necessary modifications, including concen-
tration of the analyte in the volume of the Sample solution taken. Use the Medium for preparation of the Standard solution and
dilution, if necessary, of the Sample solution.
TOLERANCES
Unless otherwise specified in the individual monograph, the requirements are met if NLT 75% of the labeled content of the
index or marker compound(s) or the extract from the units tested is dissolved in 1 h.
Unless otherwise stated in the individual monographs for dietary supplement dosage forms in this category, compliance re-
quires the testing of 6 individual units, measuring the dissolution of the dietary ingredient as the average of the 6 units tested.
PROCEDURES
Combine equal volumes of the filtered solutions of the 6 specimens withdrawn, and use the pooled sample as the Sample
solution. Determine the average amount of dietary ingredient dissolved in the pooled sample by the procedure specified in the
individual monograph. Make any necessary modifications, including concentration of the analyte in the volume of the Sample
solution taken. Use the Medium for preparation of the Standard solution and for dilution, if necessary, of the Sample solution.
TOLERANCES
Because of the diversity of chemical characteristics and solubilities of dietary ingredients pertaining to this category, general
tolerances cannot be established. See individual monographs for Tolerances.
The following tests provide limits for the permissible variations in the weights of individual tablets or capsules, expressed in
terms of the allowable deviation from the average weight of a sample. Separate procedures and limits are described herein for
capsules, uncoated tablets, and coated tablets that are intended for use as dietary supplements.
CAPSULES
Capsules meet the requirements of the following test with respect to variation in weight of contents.
Hard Capsules
Weigh 20 intact capsules individually, and determine the average weight. The requirements are met if each of the individual
weights is within the limits of 90% and 110% of the average weight.
If not all of the capsules fall within the aforementioned limits, weigh the 20 capsules individually, taking care to preserve the
identity of each capsule, and remove the contents of each capsule with the aid of a small brush or pledget of cotton. Weigh
the emptied shells individually, and calculate for each capsule the net weight of its contents by subtracting the weight of the
shell from the respective gross weight. Determine the average net content from the sum of the individual net weights. Then
determine the difference between each individual net content and the average net content: the requirements are met if (a) not
more than 2 of the differences are greater than 10% of the average net content and (b) in no case is the difference greater
than 25%.
If more than 2 but not more than 6 capsules deviate from the average between 10% and 25%, determine the net contents
of an additional 40 capsules, and determine the average content of the entire 60 capsules. Determine the 60 deviations from
the new average: the requirements are met if (a) in not more than 6 of the 60 capsules does the difference exceed 10% of the
average net content and (b) in no case does the difference exceed 25%.
Soft Capsules
Proceed as directed under Hard Capsules, but determine the net weight of the contents of individual capsules as follows.
Weigh the intact capsules individually to obtain their gross weights, taking care to preserve the identity of each capsule. Then
cut open the capsules by means of a suitable clean, dry cutting instrument, such as scissors or a sharp open blade, and remove
the contents by washing with a suitable solvent. Allow the occluded solvent to evaporate from the shells at room temperature
over a period of about 30 minutes, taking precautions to avoid uptake or loss of moisture. Weigh the individual shells, and
calculate the net contents. The requirements are as stated under Hard Capsules.
TABLETS
Weigh individually 20 whole tablets, and calculate the average weight. The requirements are met if the weights of not more
than 2 of the tablets differ from the average weight by more than the percentage listed in the accompanying table and no
tablet differs in weight by more than double that percentage.
Weigh individually 20 whole tablets, and calculate the average weight. If the coated tablets do not conform to the criteria in
the accompanying table, place 20 tablets in a beaker of water at 37°, and swirl gently for not more than 5 minutes. Examine
the cores for evidence of disintegration and repeat the procedure for a shorter time if disintegration has begun. Dry the cores
at 50° for 30 minutes. Accurately weigh 20 individual tablet cores, and calculate the average weight.
The requirements are met if the weights of not more than 2 of the tablets differ from the average weight by more than the
percentage listed in the accompanying table and no tablet differs in weight by more than double that percentage.
Criteria
Weight Variation Tolerances for Uncoated Tablets, Film-Coated Tablets, and Coated Tablets (Other Than Film-Coated Tablets)
Percentage
Average Weight of Tablet, mg Difference
130 or less 10
From 130 through 324 7.5
More than 324 5
The objective of this general chapter is to limit the amounts of elemental contaminants in finished dietary supplement dos-
age forms labeled as conforming to USP or NF standards. This general chapter is not intended to set limits for dietary ingredi-
ents. Those limits are set in the corresponding individual monographs.
The focus of this general chapter is on the four major elements of toxicological concern: arsenic, cadmium, lead, and mercu-
ry. The extent of testing can be determined using a risk-based approach that takes into account the likelihood of contamina-
tion. Manufacturers should consider the presence of unexpected elemental contaminants to determine compliance.
The levels of elemental contaminants should be restricted as shown in Table 1 unless otherwise stated in the individual mon-
ograph. Specific monographs may provide different limits for articles that need to be consumed in large quantities.
Table 1
PDE
Element (mg/day)a
Arsenic (inorganic) 15
Cadmium 5
Lead 5
Mercury (total) 15
Methylmercury (as Hg) 2
a Permitted Daily Exposure (PDE) is derived from the Provisional Tolerable Weekly Intake (PTWI) that is recommended by the Food and Agriculture Organization
of the United Nations and World Health Organization (FAO/WHO) by subtracting the daily exposure (mg/day) to each elemental contaminant from air, food,
and drinking water. A body weight of 50 kg and a safety factor are used to calculate the PDE. Other regulations (e.g., Proposition 65 in California) may require
different limits; manufacturers are responsible for compliance with applicable local requirements differing from these PDE values.
Arsenic may be measured using a nonspeciation procedure under the assumption that all arsenic contained in the supplement
is in the inorganic form. Where the limit is exceeded using a nonspeciation procedure, compliance with the limit for inorganic
arsenic shall be demonstrated on the basis of a speciation procedure. Methylmercury determination is not necessary when the
content for total mercury is less than the limit for methylmercury.
In order for a dietary supplement finished dosage form to comply with the limits for elemental contaminants as described in
this chapter, the level of elemental contaminant in the finished dietary supplement should be NMT the PDE. The following
three options are available for determining compliance with the limits for elemental contaminants in dietary supplements.
This option is generally applicable. In this option the finished dietary supplement dosage form is analyzed according to the
procedures in the general chapter Elemental Impurities—Procedures á233ñ or the speciation procedures given in this chapter.
The results obtained from the analysis of a typical serving size, scaled to a maximum daily intake, are compared to the PDE, as
stated in Table 1.
Analysis: Proceed as directed below in this chapter.
Calculate the measured amount of each elemental contaminant, in mg/daily intake, as:
Result = MVSS × N
This option is applicable to finished dietary supplement dosage forms with a maximum daily intake of NMT 10 g of the diet-
ary supplement finished product.
Analysis: Unless otherwise specified in the individual monograph, proceed with the individual ingredient as directed below
in this chapter.
Acceptance criteria: The product meets the requirements when each component used to prepare the finished dietary sup-
plement meets the limits given in Table 2.
Table 2
Individual Component Limits
Element (mg/g)a
Arsenic (inorganic)b 1.5
Cadmium 0.5
Lead 0.5
Mercury (total) 1.5
Methylmercury (as Hg)c 0.2
a The limits for individual components are based on a maximum daily intake of 10 g of a dietary supplement and are intended for use only with Options for
Compliance with the Limits of Elemental Contaminants, Individual Component Option.
b Arsenic may be measured using a nonspeciation procedure under the assumption that all arsenic contained in the supplement is in the inorganic form. Where
the limit is exceeded using a nonspeciation procedure, compliance with the limit for inorganic arsenic shall be demonstrated on the basis of a speciation proce-
dure.
c Methylmercury determination is not necessary when the content for total mercury is less than the limit for methylmercury.
[NOTE—If all components in a formulation meet the limits given for the Individual Component Limits, these components can be
used in any proportion. No further calculation is necessary.]
Summation Option
This option can be used for finished dietary supplement dosage forms that are consumed in quantities greater than 10 g/day
or where the acceptance limit for any contaminant in any component of the dietary supplement exceeds the applicable Indi-
vidual Component Limits.
Analysis: Unless otherwise specified in the individual monograph, proceed with the individual ingredient as directed below
in this chapter.
Calculate the amount of each elemental contaminant, in mg/daily intake, present in the finished dietary supplement dosage
form:
Result = S(Ci × Wi) × N
Performance-based methodology for analysis of total elemental contaminants in general chapter Elemental Impurities—Proce-
dures á233ñ is applicable for dietary supplements. The validation necessity will vary depending on the situation. In all three op-
tions described in the section Options for Compliance with the Limits of Elemental Contaminants, the use of Validation of Limit
Procedures (see Elemental Impurities—Procedures á233ñ) may be appropriate. However, for the Summation Option, acceptable
levels of validation must be determined on a case-by-case basis. Validation of a procedure using the Validation of Quantitative
Procedures (see Elemental Impurities—Procedures á233ñ) is acceptable for all options under all circumstances and is generally pre-
ferred. The determination of the level of validation necessity is at the discretion of the manufacturer and the competent regula-
tory authority.
Where the level of total arsenic exceeds the limit recommended in this chapter, speciation may be used to determine the
amount of inorganic arsenic present. The following procedure is suggested for determination of inorganic arsenic, but any vali-
dated procedure shown to give equivalent or better results can be used.
Apparatus
See Figure 1.
Figure 1. Special apparatus for the determination of inorganic arsenic (A, 250-mL distillation flask; B, receiver chamber, ap-
proximately 50-mL capacity; C, reflux condenser; D, splash head or security funnel).
Reagents
Distillation-reducing solution: 30.0 g of potassium iodide in 100 mL of water. [NOTE—Prepare fresh on the day of use.]
Control: 6.0 mg of arsenic (As) (6.0 mL of Standard Arsenic Solution). [NOTE—Use this amount rather than the 3.0 mL
specified for Standard Preparation in the general chapter Arsenic á211ñ, Method I.]
Sample solution: Transfer a 2.00-g sample that has previously been ground to pass through a 60-mesh screen to a distil-
lation flask (A). To the flask add 35 mL of hydrochloric acid 6.6 N and 15 mL of Distillation-reducing solution, let stand for 5 min
to ensure reduction of arsenic [As(V)], connect the flask to the receiver chamber (B), complete the assembly of the apparatus,
and begin circulating tap water through the condenser (C). Half-fill the lower two bulbs of the splash head (D) with water.
Maneuver the stopcock to cause the contents of the receiver chamber to drain into the distillation flask, heat the flask until the
temperature above the solution reaches 100°–110°, and continue refluxing at this temperature for 15 min. Close the stopcock,
continue heating at 108°–110°, and collect 30–33 mL of distillate in the receiver chamber. Remove the heating source, and
allow the temperature to drop to about 80°.
Close the stopcock, and add a second 35-mL portion of 6.6 N hydrochloride (HCl) and 15 mL of the Distillation-reducing
solution through the thermometer opening to the distillation flask. Replace the thermometer, increase the temperature to
100°–110°, and collect a second 30- to 33-mL portion of distillate in the receiver chamber. Drain the second distillate into the
beaker containing the first portion, and cool the combined distillate to room temperature. Remove the splash head, and wash
its contents into the beaker. Also, wash down the inside of the condenser and receiver chamber with water, collecting the
washings into the beaker. Transfer to a 100-mL volumetric flask, and complete with water to volume.
Analysis: Determine the arsenic content by the ICP-MS procedure in Elemental Impurities—Procedures á233ñ. Alternatively,
add 2 mL of potassium iodide TS and 0.5 mL of stronger acid stannous chloride TS to the Sample solution contained in the
Erlenmeyer flask, and proceed as directed in Arsenic á211ñ, Method I, Procedure, beginning with “Allow to stand at room tem-
perature for 30 minutes.”
Where methylmercury determination is required, the following procedure is suggested. However, any validated procedure
shown to give equivalent or better results can be used.
Procedure 1
This procedure uses an aqueous extraction of the mercury species with an L-cysteine solution, HPLC separation of the deriv-
atized mercury species with a mobile phase also containing L-cysteine, and ICP-MS detection.
Cysteine solution: 1% L-cysteine hydrochloride monohydrate in water
Mobile phase: 0.1% L-cysteine hydrochloride monohydrate and 0.1% L-cysteine free base in water
Standard stock solution: 1 mg/mL of mercury (Hg) as methylmercury chloride, and 1 mg of mercury (Hg) as mercury
chloride in 5% hydrochloric acid containing 0.2 mg/mL of L-cysteine hydrochloride monohydrate
Standard solution: 2 ng/mL of mercury as methylmercury from Standard stock solution in Cysteine solution
Sample solutions
For supplements in tablet form: Weigh, and finely powder a counted number of tablets. Transfer an accurately weighed
portion of the powder equivalent to 0.5 times the daily recommended intake to a tared vial. Add 50.0 mL of Cysteine solution
accurately weighed, cap the vial, and shake vigorously. Place the vial in a water bath at 60° for 60 min. Shake the vial again,
and return to the water bath for another period of 60 min. Shake the vial again, and allow to cool at room temperature for
about 20 min. Filter through a 0.45-mm polyethylene membrane. [NOTE—Prepare fresh on the day of use.]
For supplements in capsule form: Weigh accurately NLT 20 capsules, and determine the average weight. Place a num-
ber of capsules equivalent to about 5 times the daily recommended intake in a blender, add 500.0 mL of Cysteine solution and
blend to obtain a homogenous analytical suspension. Transfer 50.0 mL of this analytical suspension to a vial, cap the vial, and
shake vigorously. Place the vial in a water bath at 60° for 60 min. Shake the vial again, and return to the water bath for anoth-
er period of 60 min. Shake the vial again, and allow to cool at room temperature for about 20 min. Filter through a 0.45-mm
polyethylene membrane. [NOTE—Prepare fresh on the day of use.]
For supplements in liquid form: Weigh accurately an amount of the liquid equivalent to 0.5 times the daily recommen-
ded intake into a vial, and add 50.0 mL of Cysteine solution. Cap the vial, and shake vigorously. Place the vial in a water bath at
60° for 60 min. Shake the vial again, and return to the water bath for another period of 60 min. Shake the vial again, and
allow to cool at room temperature for about 20 min. Filter through a 0.45-mm polyethylene membrane. [NOTE—Prepare fresh
on the day of use.]
Chromatographic system
(See Chromatography á621ñ, System Suitability.)
Mode: LC
Detector: ICP-MS at a mass-to-charge ratio of 202
Column: 4.6-mm × 15-cm; 4-mm packing L1
Flow rate: 1 mL/min
Injection volume: 50 mL
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 3 between the peak representing mercury (Hg2+) species at a relative retention time of about 0.56 and
the peak representing methylmercury (MeHg+) species at a relative retention time of 1.0
Relative standard deviation: NMT 10.0%, Standard solution
Analysis
Samples: Standard solution and the appropriate Sample solution
Determine the peak area of the peak representative of methylmercury in the chromatograms of the Sample solution and
Standard solution.
Acceptance criteria: 0.2 mg/daily intake: The peak area for methylmercury in the chromatogram of the Sample solution is
NMT the peak area for methylmercury in the chromatogram of the Standard solution.
INTRODUCTION
Federal regulations do not permit irradiation of dietary ingredients or dietary supplements for sanitation purposes. Under
section 201(s) of the Federal Food, Drug, and Cosmetic Act [21 USC 321(s)], irradiation is considered an additive, and as such
it requires FDA approval. Foods that are irradiated should be adequately labeled according to international and national guide-
lines with statements such as “Treated with radiation” or “Treated by irradiation” in addition to information required by other
regulations, including the irradiation logo, the Radura [21 USC 321(s)]. Overexposure to irradiation may have negative effects
on product quality. Currently, several independent methods are used to identify irradiated foodstuffs, including dietary supple-
ments. These methods have been validated and are recognized worldwide.
Procedures based on luminescence are widely applied and include screening by photostimulated luminescence (PSL), which
is a rapid, simple preliminary screening method to detect irradiation, and a subsequent thermoluminescence (TL) procedure to
confirm that the sample has been irradiated. PSL is less time consuming than TL because the inorganic/silicate mineral source
of luminescence does not need to be isolated from any organic components present. Both PSL and TL signal intensities are
affected by irradiation dose as well as by the nature and amount of inorganic material. TL analysis is one of the detection
methods used for confirming the presence of irradiated foods, herbs, spices, vegetables, and fruits, although it has certain limi-
tations: for example, samples must contain sufficient amounts of silicates that can be successfully separated from the samples.
The lengthy preparation and need for irradiation in all cases limits TL to a small number of laboratories.
The procedures described in this chapter can be used both by regulatory authorities and by producers and suppliers of
foods, including dietary supplements, to detect undeclared irradiated products in the market for purposes of determining
compliance with regulations.
Most of the natural dietary ingredients that are either cultivated or wild contain silicate minerals, calcite, or hydroxyapatite.
Exposure to ionizing radiation from gamma rays (60Co or 137Cs), electron beams (up to 10 MeV), or x-rays (up to 5 MeV) stimu-
lates electrons in those types of crystals, resulting in energized electrons being stored in the crystal lattice. Trapped high-ener-
gy electrons can be released by stimulation with light or controlled heat, leaving electron holes in the crystal lattice. The ener-
gy thus released is detected as luminescence. PSL occurs if light is applied to the system, and TL occurs when heat is applied to
the system. The recorded luminescence intensity is proportional to the initial radiation dose absorbed.
Samples for both PSL and TL measurements must be taken from a light-protected position of the bulk samples, and expo-
sure to high temperature and light must be avoided after the samples are taken from the bulk. Sample preparation and subse-
quent PSL or TL measurement must be conducted under subdued lighting conditions when possible.
TL has been widely used for detection of irradiation in food and dietary ingredients from which silicate minerals can be isola-
ted. In this chapter, PSL and TL are described as the most appropriate detection methods for the materials typically used as
dietary ingredients. The two methods are both radiation-specific phenomena, based on the observation that photon counts of
irradiated samples usually are higher than those of nonirradiated samples. However, all available methods have some limita-
tions in terms of their specific application range, product-to-product variation, complexity of or interference from the food ma-
trix, and low concentration of radiation-induced markers.
PSL, also known as optically stimulated luminescence, is based on the emission of light in the 300–600 nm range from irra-
diated samples when illuminated at longer wavelengths in the near-IR region. PSL analysis allows multiple measurements to be
performed without sample pretreatment in a short period of time. PSL is also a nondestructive method that does not require
separation of inorganic minerals and organic compounds. PSL is based on optical stimulation of mineral debris, typically sili-
cates, and bioinorganic materials such as calcite, feldspar, or hydroxyapatite, and its sensitivity depends on the quantities and
types of minerals present in the sample. Before the measurement of PSL, two thresholds are set: the lower typically is set at
700 counts/min (T1), and the upper typically is set at 5000 counts/min (T2). These two thresholds serve to classify the sam-
ples. After initial screening of PSL intensity from the samples is performed, the results are classified into negative (counts/ min
less than T1), intermediate (counts/min NLT T1 and NMT T2), or positive (counts/min more than T2). A second measurement
after a known irradiation dose, known as calibrated PSL (CalPSL), is applied in cases of poorly defined sample matrices whose
luminescence sensitivities are not well established. By comparing the screening PSL measurement against its CalPSL response,
analysts can take into account variation in detection sensitivity due to variable amounts and different types of minerals present
in a given sample. CalPSL measurements are recommended to rule out false negative results due to low mineral content.
INSTRUMENTATION
The PSL system consists of pulsed IR sources for photostimulation, a single-photon counting system for highly sensitive de-
tection of luminescence, a sample chamber, and a computer for data analysis.
The instrumental setup procedure includes checks of irradiated and nonirradiated materials, as well as establishing measure-
ment parameters (cycle time, thresholds, and data-recording conditions). Measurement of initial background counts and peri-
odic measurement of counts in the empty chamber should be conducted in subdued lighting to confirm lack of instrument
contamination. The PSL signals (photon counts) emitted from the sample/second are automatically accumulated in the com-
puter and are reported as counts/min. [NOTE—All experiments should be conducted under subdued lighting. In order to mini-
mize the risk of cross-contamination, all preparations are performed in a laminar-flow cabinet.]
Procedure
PSL screening—Dispense and weigh two portions of the sample (5–10 g, depending on the density of the product) into two
separate 50-mm disposable Petri dishes to cover the Petri base in a thin layer. Dispense samples in subdued lighting to mini-
mize bleaching and under a laminar-flow cabinet to minimize cross-contamination. Measure the PSL of the sample for 1 min
on the duplicate aliquots, and calculate the mean.
Calibrated PSL (CalPSL)—[NOTE—CalPSL testing may be done only once as part of the validation of the procedure for each ma-
terial.]
Dispense and weigh two portions of the sample (5–10 g, depending on the density of the product) into 50-mm disposable
Petri dishes to cover the Petri base in a thin layer. Measure the PSL of the sample for 1 min on duplicate aliquots, and for each
sample calculate the mean. Irradiate the sample to a known dose of 1 kGy after the initial screening. Repeat the PSL measure-
ment (CalPSL).
Evaluation—Classify results in the following manner:
<T1: negative, no evidence of PSL
>T1 and <T2: intermediate, weak PSL signal
>T2: positive, stronger signal.
Acceptance criteria—Nonirradiated samples produce negative results in the PSL screening and are known to produce positive
results in the CalPSL. [NOTE—Negative signals in the PSL screening are generally associated with nonirradiated material but can
result from low-sensitivity irradiated materials. To evaluate whether a sample is a low-sensitivity material, assessment of the
signals using the CalPSL option is necessary. Positive signals in the PSL screening are associated with irradiated material. Sam-
ples that are classified as intermediate require further investigation by the TL method to determine their irradiation status.]
The TL analysis is based on physical changes in silicate minerals that are present in many food samples and dietary ingredi-
ents. The silicate minerals are able to store the absorbed radiation energy. A TL reader measures the amount of light that is
emitted during controlled heating. The TL of the sample (TL1) is compared to that of the same sample following irradiation at
1 kGy (TL2). If the TL ratio (TL1/TL2) is greater than 0.1, the sample is considered to be irradiated. In order to use the TL method
for the detection of irradiated foods or dietary ingredients, silicate minerals must be isolated from the samples.
INSTRUMENTATION
TL measurements can be performed with a TL detector that meets the specifications in Table 1.
Table 1. Thermoluminesence Detector Specificationsa
Photon: energies >5 keV
Neutron: thermal to 100 MeV
Radiationb Electron/beta: energies >70 keV
10 mGy to 1 Gy (1 mrad to 100 rad) linear
Measurement ranges 1 Gy to 20 Gy (100 rad to 2000 rad) supralinear
For 1 mGy (100 mrad) 137Cs doses, <2% standard deviation of 10 sequen-
Repeatability tial measurements
50° to approximately 500°,
Heating plate approximately 6°/s
a Harshaw TLD 3500 (Thermo Fisher Scientific, Waltham, MA) and Nanogray TL2000 (Nanogray, Osaka, Japan) are suitable.
b Gamma rays from 60Co or 10-MeV electron beams are suitable radiation sources.
Sodium polytungstate solution: Prepare a solution of sodium polytungstate in water with a final density of 2 g/mL.
Mineral extraction: Suspend 3–20 g of sample with 50–100 mL of pure water, and sonicate for about 5 min. Sieve through
a 250-mm nylon mesh, rinsing the mesh with water each time by using a strong jet from a wash bottle into a larger beaker
(500–1000 mL). Allow the minerals to settle for 5 min and decant most of the water, leaving the minerals in <50 mL of water.
Transfer the mineral fraction to a centrifuge tube, and centrifuge for 1 min at 1000 × g. Discard the water layer.
Preconcentration density separation: Add 5 mL of the Sodium polytungstate solution to the minerals in the centrifuge tube.
Shake or mix on a vortex mixer, then sonicate for 5–15 min. Centrifuge for 2 min at 1000 × g. Silicate minerals (density 2.5–
2.7 g/mL) precipitate, whereas organic components float. Carefully overlay a layer of water on the Sodium polytungstate solu-
tion, and discard the water layer and the organic materials by decantation or vacuum suction, leaving the minerals behind in
the polytungstate layer. Carefully remove the Sodium polytungstate solution layer, and wash the minerals twice with water (cen-
trifuge at 1000 × g briefly). Add 1–2 mL of 1 M hydrochloric acid, shake, and leave for 10 min in the dark to dissolve carbo-
nates adhering to the silicate materials. Neutralize the acid with 1 M ammonium hydroxide. Fill the tube with water. Allow the
minerals to settle, and centrifuge. Discard the water, and wash the minerals twice with water.
Fixing the minerals on disc for TL measurement: Add 3 mL of acetone to the preconcentrated minerals, and shake to dis-
place residual water. Use a stainless steel disc suitable for the TL reader in use. [NOTE—Discs typically are 9–10 mm in diameter
and 0.25–0.50 mm in thickness]. Carefully clean the disc by rinsing in water, wash two to three times with acetone, and dry in
an oven. Store under dust-free conditions. Record the weight of the clean disc immediately before use. Transfer the minerals
(in acetone), and dry the disc at 50° overnight (lab oven). Weigh the disc, and calculate the mass of minerals. Repeat the ex-
traction, if necessary, to meet the system suitability requirements. [NOTE—The mineral sample amount is typically between 0.1
mg and 5 mg]. The deposited minerals can be fixed on the disc by using silicone spray (or by layering with 0.2% carboxyme-
thylcellulose) and drying at 50° overnight.
Blank discs: Use clean discs.
TL MEASUREMENT
TL measurements are performed using a TL reader. Register the TL emission as a function of temperature (glow curves).
Minimum detectable integrated TL intensity level (MDL): Integrate the TL intensity of the first glow of the blank discs,
and calculate the standard deviation. The MDL is three times the standard deviation of the integrated TL intensity of the blank
discs.
System suitability: The integrated TL intensity of the irradiated sample (TL2) should be at least 10 times the MDL.
Measurement of TL1
First-glow TL1 measurement—Set the instrument to an initial temperature of 70°, a heating rate of 6°/s, and a final temperature
of 350°. Flush the chamber with nitrogen. Place the sample disc on the heating plate of the TL reader, and measure the glow
curve. Determine TL1 as the integrated TL signal between 150° and 250°. Measure the background glow for the sample (BG1)
after cooling the sample to 50°. After measuring TL1 and BG1, measure the weight of the sample (BW1).
Irradiation—Irradiate the disc with the minerals, with a defined radiation dose of about 1 kGy. [NOTE—A source of 60Co gamma
rays is suitable.] Store at 50° overnight.
Measurement of TL2
Second-glow TL2 measurement—Measure the TL for the irradiated sample (TL2) as described for TL1. Measure the background
glow for the irradiated sample (BG2) after cooling the sample to 50°, and record the weight of the sample plate with the sam-
ple (BW2). Measure the blank levels for process control (without the sample), following the procedures at each stage. Calculate
the MDL as the integrated TL intensity of the blank plus three standard deviations.
TL glow ratio: The TL glow ratio per sample weight is calculated as described below:
TL glow ratio = [(TL1 − BG1)/BW1]/[(TL2 − BG2)/BW2]
GENERAL PROVISIONS
The principles included in this chapter contain recommended minimum current good manufacturing practices for the meth-
ods to be used in, and the facilities and controls to be used for, the manufacture, holding, packaging, labeling, and distribu-
tion of dietary ingredients and dietary supplements. These principles are set forth to ensure that such products meet the re-
quirements of safety, have the identity and strength, and meet the quality and purity characteristics that they are represented
to possess.
Excluded from this chapter are establishments engaged solely in the harvesting, storage, or distribution of one or more “raw
agricultural commodities” as defined in Section 201(r) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 321(r)), which
are ordinarily cleaned, prepared, treated, or otherwise processed before being marketed to the consuming public.
The requirements pertaining to holding dietary ingredients and dietary supplements do not apply to holding those dietary
supplements at a retail establishment for the sole purpose of direct retail sale to individual consumers. A retail establishment
does not include a warehouse or other storage facility for a retailer or a warehouse or other storage facility that sells directly to
individual consumers.
A glossary of terms used in this chapter is presented at the end.
A quality control unit shall be established that has the responsibility and authority to approve or reject all raw materials,
product containers, closures, in-process materials, packaging material, labeling, and finished dietary supplements, and the au-
thority to review production records to ensure that no errors have occurred or, if errors have occurred, that they have been
fully investigated. The quality control unit should be responsible for approving or rejecting products manufactured, processed,
packed, or held under contract by another company.
Adequate laboratory facilities for the testing and approval (or rejection) of raw materials, product containers, closures, pack-
aging materials, in-process materials, dietary ingredients, and dietary supplements should be available to the quality control
unit.
The quality control unit should have the responsibility for approving or rejecting all procedures or specifications that impact
on the identity, strength, quality, and purity of the dietary supplement. All responsibilities and procedures applicable to the
quality control unit shall be in writing.
The designated person within the Quality Control Unit who conducts a material review and makes the disposition decision
must, at the time of performance, document that material review and disposition decision.
Personnel Qualifications
Each person engaged in the manufacture of dietary ingredients and dietary supplements should have the proper education,
training, and experience (or any combination thereof) needed to perform the assigned functions. Training should be in the
particular operation(s) that the employee performs as they relate to the employee's functions.
Appropriate documentation of training shall be retained by the company.
Each person responsible for supervising the manufacture of a dietary ingredient, a dietary supplement, or both should have
the proper education, training, and experience (or any combination thereof) to perform assigned functions in such a manner
as to provide assurance that the product has the safety, identity, strength, quality, and purity that it is represented to possess.
An adequate number of qualified personnel to perform and supervise the manufacture of each dietary ingredient, dietary
supplement, or both product should be provided.
Personnel Responsibilities
The company management shall take all reasonable measures and precautions to ensure the following:
1. Disease control. Any person who, by medical examination or supervisory observation, is shown to have, or appears to
have, an illness, open lesion, including boils, sores, or infected wounds, or any other abnormal source of microbial con-
tamination by which there is a reasonable possibility of an in-process or finished dietary ingredient or dietary supplement
becoming adulterated, or processing equipment, utensils, or packaging materials becoming contaminated, shall be exclu-
ded from any operations which may be expected to result in such adulteration or contamination until the condition is
corrected. Personnel shall be instructed to report such health conditions to their supervisors.
2. Cleanliness. All persons working in direct contact with raw materials, in-process or finished dietary ingredients and dietary
supplements, processing equipment, utensils, or packaging materials shall conform to hygienic practices while on duty to
the extent necessary to protect against adulteration or contamination of such materials. The methods for maintaining
cleanliness include, but are not limited to, the following:
— Wearing outer garments suitable to the operation in a manner that protects against the adulteration of raw materials
or of in-process or finished dietary ingredients and dietary supplements, or contamination of processing equipment,
utensils, or packaging materials;
— Maintaining adequate personal cleanliness;
— Removing cosmetics from parts of the body that may contact raw materials, in-process or finished dietary ingredients
and dietary supplements, equipment, utensils, or containers;
— Washing hands thoroughly (and sanitizing if necessary to protect against contamination with undesirable microor-
ganisms) in an adequate hand-washing facility before starting work, after each absence from the work station, and at
any other time when the hands may have become soiled or contaminated;
— Removing all unsecured jewelry and other objects that might fall into raw materials, in-process or finished dietary
ingredients and dietary supplements, equipment, or containers, and removing hand jewelry that cannot be ade-
quately sanitized during periods in which in-process or finished product is manipulated by hand. If such hand jewelry
and cosmetics cannot be removed, they may be covered by material that can be maintained in an intact, clean, and
sanitary condition and that effectively protects against the adulteration of dietary ingredients and dietary supple-
ments or contamination of processing equipment, utensils or packaging materials;
— Maintaining gloves, if they are used in raw materials or in in-process or finished product handling, in an intact, clean,
and sanitary condition. The gloves should be of a material that adequately protects the product from contamination;
— Wearing, where appropriate, in an effective manner, hair nets, caps, beard covers, or other effective hair restraints;
— Storing clothing or other personal belongings in areas other than where in-process or finished product is exposed or
where processing equipment or utensils are washed;
— Confining the following actions to areas other than where in-process or finished product may be stored or exposed,
or where processing equipment or utensils are washed: eating food, chewing gum, drinking beverages, or using to-
bacco; and
— Taking any other necessary precautions to protect against adulteration of raw materials or of in-process or finished
product, or contamination of processing equipment, utensils, or packaging materials with microorganisms or foreign
substances, including, but not limited to, perspiration, hair, cosmetics, tobacco, chemicals, and medicines applied to
the skin.
Grounds
The grounds about a dietary ingredient manufacturing plant and a dietary supplement manufacturing plant under the con-
trol of the operator shall be kept in a condition that will protect against the adulteration of dietary ingredients and dietary
supplements. The methods for adequate maintenance of grounds include, but are not limited to, the following:
— Properly storing equipment, removing litter and waste, and cutting weeds or grass within the immediate vicinity of the
plant building or structures that may constitute an attractant, breeding place, or harborage for pests;
— Maintaining roads, yards, and parking lots so that they do not constitute a source of adulteration in areas where product
is exposed;
— Adequately draining areas that may contribute to product adulteration by seepage, foot-borne filth, or providing a breed-
ing place for pests; and
— Operating systems for waste treatment and disposal in an adequate manner so that they do not constitute a source of
adulteration in areas where product is exposed. If the plant grounds are bordered by grounds not under the operator's
control and not maintained in the manner described above, care shall be exercised in the plant by inspection, extermina-
tion, or other means to exclude pests, dirt, and filth that may be a source of product adulteration.
Building Design
Any building or buildings used in the manufacture of a dietary ingredient, a dietary supplement, or both should be of suita-
ble size and shall be constructed in such a manner that floors, walls, and ceilings may be adequately cleaned and kept clean
and in good repair; that drips or condensates from fixtures, ducts, and pipes do not adulterate raw materials or in-process or
finished dietary ingredients and dietary supplements, or contaminate product containers, utensils, or packaging materials; and
that aisles or working spaces are provided between equipment and walls and are adequately unobstructed and of adequate
width to permit employees to perform their duties and to protect against adulterating in-process or finished product, or con-
taminating processing equipment with clothing or personal contact. Adequate screening or other protection against pests and
insects should be installed, where necessary. The building should have adequate space for the orderly placement of equipment
and materials to prevent mixups between different raw materials, product containers, closures, labeling, in-process materials,
or finished products, and to prevent contamination. The flow of raw materials, product containers, closures, labeling, in-proc-
ess materials, and products through the building or buildings should be designed to prevent contamination.
Operations should be performed within specifically defined areas of adequate size to prevent contamination or mixups or
adulteration of in-process or finished dietary ingredients and dietary supplements, or contamination of processing equipment,
utensils, or packaging materials with microorganisms, chemicals, filth, or other extraneous materials. The potential for mixups
and product adulteration may be reduced by adequate product safety controls and operating practices or effective design, in-
cluding the separation of operations in which contamination is likely to occur, by one or more of the following means: loca-
tion, time, partition, airflow, enclosed systems, or other effective means. There should be separate or defined areas as follows:
1. An area for the receipt, identification, storage, and withholding from use of components, product containers, closures,
and labeling, pending the appropriate sampling, testing, or examination by the quality control unit before release for
manufacturing or packaging;
2. An area for the storage of released components, product containers, closures, and labeling;
3. An area for storage of in-process materials;
4. An area for manufacturing and processing operations;
5. An area for packaging and labeling operations; and
6. An area for control and laboratory operations.
Any building used in the manufacture of a dietary ingredient or a dietary supplement shall permit the taking of proper pre-
cautions to protect dietary ingredients or dietary supplements in outdoor bulk fermentation vessels by any effective means,
including the following:
(i) Using protective coverings,
(ii) Controlling areas over and around the vessels to eliminate harborages for pests,
Official text. Reprinted from First Supplement to USP38-NF33.
830 á2750ñ Manufacturing Practices / Dietary Supplements DSC
(iii) Checking on a regular basis for pests and pest infestation, and
(iv) Skimming the fermentation vessels, as necessary.
Lighting
Adequate lighting shall be provided in all areas and should not expose bulk or finished product to adulteration or contami-
nation. Adequate lighting should be provided in hand-washing areas, dressing and locker rooms, and toilet rooms, and in all
areas where product is examined, processed, or stored and where equipment or utensils are cleaned; and such lighting should
provide safety-type light bulbs, fixtures, skylights, or other glass suspended over exposed product in any step of preparation or
otherwise protect against product adulteration in case of glass breakage.
Adequate ventilation shall be provided, as well as equipment for adequate control over microorganisms, dust, humidity, and
temperature when used in the manufacture of a dietary ingredient and a dietary supplement to minimize odors and vapors
(including steam and noxious fumes) in areas where they may adulterate dietary ingredients and dietary supplements; and to
locate and operate fans and other air-blowing equipment in a manner that minimizes the potential for adulterating raw mate-
rials, in-process or finished dietary ingredients and dietary supplements, or contaminating processing equipment, utensils, or
packaging materials.
Plumbing
The plumbing in the physical plant must be of an adequate size and design and be adequately installed and maintained to:
— Carry sufficient amounts of water to the required locations throughout the physical plant;
— Properly convey sewage and liquid disposable waste from the physical plant; and
— Avoid being a source of contamination to components, raw materials, dietary ingredients, dietary supplements, water
supplies, or any contact surface, or creating an unsanitary condition.
Potable water at a suitable temperature, and under pressure as needed, should be supplied in a plumbing system free of
defects that could contribute contamination to any dietary ingredients and dietary supplements. Potable water should meet
the standards prescribed in the Environmental Protection Agency's Primary Drinking Water Regulations (40 CFR Part 141) or
any state or local drinking water requirements that are more stringent. Water not meeting such standards should not be per-
mitted in the potable water system for Purified Water. If potable water is to be used as a raw material, it should be further
purified to satisfy compendial requirements.
Drains should be of adequate size and, where connected directly to a sewer, should have an air break or other mechanical
device to prevent back-siphonage.
Sewage, trash, and other refuse in and from the building and immediate premises shall be disposed of in a safe and sanitary
manner.
Adequate washing facilities shall be provided, including hot and cold water, soap or detergent, air driers or single-service
towels, and clean toilet facilities easily accessible to working areas.
Any building used in the manufacture of a dietary ingredient, a dietary supplement, or both should be maintained in a clean
and sanitary condition and shall be kept in repair sufficient to prevent raw materials and in-process or finished dietary ingredi-
ents and dietary supplements from becoming adulterated. It shall be free of infestation by rodents, birds, insects, and other
vermin. Trash and organic waste matter shall be held and disposed of in a timely and sanitary manner.
Cleaning compounds and sanitizing agents used in cleaning and sanitizing procedures shall be free from undesirable micro-
organisms and shall be safe and adequate under the conditions of use. Compliance with this requirement may be verified by
any effective means, including purchase of these substances under a supplier's guarantee or certification, or examination of
these substances for contamination. Only the following toxic materials may be used or stored in a plant where product is pro-
cessed or exposed:
1. Those required to maintain clean and sanitary conditions;
2. Those necessary for use in laboratory testing procedures;
3. Those necessary for plant and equipment maintenance and operation; and
Equipment and utensils used in the manufacture of dietary ingredients and dietary supplements shall be of appropriate de-
sign or selection, adequate size, and suitably located to facilitate operations for its intended use and for its cleaning and main-
tenance and to ensure that the specifications of dietary ingredients and dietary supplements are correct and are met.
Equipment and utensils include, but are not limited to, the following:
— Equipment used to hold or convey;
— Equipment used to measure;
— Equipment using compressed air or gas;
— Equipment used to carry out processes in closed pipes and vessels; and
— Equipment used in automatic, mechanical, or electronic systems.
Construction
Instruments or controls used in the manufacturing, packaging, labeling, or holding of a dietary ingredient, a dietary supple-
ment, or both; and instruments or controls that are used to measure, regulate, or record temperatures, hydrogen-ion concen-
tration (pH), water activity, or other conditions, and to control or prevent the growth of microorganisms or other contamina-
tion must be:
— Accurate and precise,
— Adequately maintained, and
— Adequate in number for their designated uses.
For any automated, mechanical, or electronic equipment that is used to manufacture, package, label, or hold a dietary in-
gredient, a dietary supplement, or both:
— The suitability of the equipment must be determined by ensuring that the equipment is capable of operating satisfactorily
within the operating limits required by the process;
— The equipment must be routinely calibrated, inspected, or checked to ensure proper performance. The quality control
unit must approve these calibrations, inspections, or checks;
— The appropriate controls for automated, mechanical, and electronic equipment (including software for a computer-con-
trolled process) must be established and used to ensure that any changes to the manufacturing, packaging, labeling,
holding, or other operations are approved by the quality control unit and instituted only by authorized personnel; and
— The appropriate controls must be established and used to ensure that the equipment functions in accordance with its in-
tended use. These controls must be approved by the quality control unit.
Compressed air or other gases introduced mechanically into or onto raw materials, in-process materials, dietary ingredients,
dietary supplements, or contact surfaces, or that are used to clean any contact surface, must be treated in such a way that the
raw material, in-process material, dietary ingredient, dietary supplement, or contact surface is not contaminated.
Equipment and utensils shall be cleaned, maintained, and sanitized at adequate intervals, between the manufacture of differ-
ent batches of the same product and between the manufacture of different products, to prevent malfunctions or contamina-
tion that would alter the safety, identity, strength, quality, or purity of the product beyond the established requirements.
In wet processing during manufacturing, all contact surfaces must be cleaned and sanitized, as necessary, to protect against
the introduction of microorganisms into components, dietary ingredients, or dietary supplements. When cleaning and sanitiz-
ing is necessary, all contact surfaces must be cleaned and sanitized before use and after any interruption during which the
contact surface may have become contaminated.
In a continuous production operation or in back-to-back consecutive operations, which involve different batches of the same
dietary ingredient or dietary supplement, the contact surfaces must be adequately cleaned and sanitized.
The surfaces that do not come into direct contact with raw materials, in-process materials, dietary ingredients, or dietary
supplements must be cleaned as frequently as necessary to protect against contaminating raw materials, in-process materials,
dietary ingredients, or dietary supplements.
Single-service articles (such as utensils intended for one-time use, paper cups, and paper towels) must be:
— Stored in appropriate containers; and
— Handled, dispensed, used, and disposed of in a manner that protects against contamination of raw materials, in-process
materials, dietary ingredients, dietary supplements, or any contact surface.
Cleaning compounds and sanitizing agents must be adequate for their intended use and safe under their conditions of use.
The portable equipment and utensils that have contact surfaces must be cleaned, sanitized, and then stored in a location
and manner that protects them from contamination.
Written procedures for cleaning and maintaining equipment, including utensils, used in the manufacture of a product
should be established and followed. These procedures should include, but are not necessarily limited to, the following:
— Assignment of responsibility for cleaning and maintaining equipment;
— Maintenance and cleaning schedules, including, where adequate, sanitizing schedules;
— A description in sufficient detail of the methods, equipment, and materials used in cleaning and maintenance operations,
and the methods of disassembling and reassembling equipment, as necessary, to ensure proper cleaning and mainte-
nance;
— Removal or obliteration of previous batch identification;
— Identification and protection of clean equipment from contamination before use;
— Inspection of equipment for cleanliness immediately before use;
— Regular calibration and inspection of equipment, or checking machines, to ensure proper performance and function must
be conducted:
(a) Before first use; and
(b) At frequency specified in writing by supporting references.
— Instruments or controls that cannot be adjusted to agree with the reference standard must be repaired or replaced.
A written record of calibration, inspection, maintenance of equipment, and major equipment cleaning and use shall be
maintained in individual equipment logs that show the date, product, and lot number of each batch processed. The persons
performing the cleaning shall record in the log that the work was performed. Entries in the log should be in chronological
order.
The following is specified in order to keep records related to automated, or electric equipment:
— There must be backup file(s) of current software programs (and of outdated software that is necessary to retrieve records
that are required to be retained, in accordance with the section Records and Reports in this chapter, when current software
is not able to retrieve such records) and of data entered into computer systems used to manufacture, package, label, or
hold dietary supplements.
(a) A backup file (e.g., a hard copy of data entered, diskettes, tapes, microfilm, or compact disks) must be an exact and
complete record of the data entered.
(b) Backup software programs and data must be kept secure from alterations, inadvertent erasures, or loss.
Written procedures describing in sufficient detail the receipt, identification, storage, handling, sampling, testing, and appro-
val or rejection of raw materials, product containers, and closures should be provided.
Raw materials, product containers, and closures at all times should be handled and stored in a manner to prevent contami-
nation.
Raw agricultural materials that contain soil or other contaminants shall be washed or cleaned as necessary. Water used for
washing, rinsing, or conveying raw agricultural materials shall be safe and of adequate sanitary quality. Notwithstanding the
general requirement for potable water, water may be reused for washing, rinsing, or conveying raw agricultural materials if it
does not increase the level of contamination of such materials.
Bagged or boxed raw materials of product containers or closures should be stored off the floor and suitably spaced to permit
cleaning and inspection.
Each lot should be appropriately identified as to its status (i.e., quarantined, approved, or rejected).
Receipt and Storage of Untested Raw Materials, Product Containers, and Closures
Written procedures shall be established and followed describing the receipt, identification, examination, handling, and sam-
pling of raw materials. Upon receipt and before acceptance, each container or grouping of containers of raw materials, prod-
uct containers, and closures should be examined visually for appropriate labeling as to contents, container damage, or broken
seals, and for contamination. They are then stored under quarantine until they have been tested or examined, as appropriate,
and released.
Raw materials shall be held in bulk, or in containers designed and constructed so as to protect against adulteration, and shall
be held at such temperature and relative humidity and in such a manner as to prevent a dietary ingredient or dietary supple-
ment from becoming adulterated. Frozen raw materials and other ingredients shall be kept frozen. If thawing is required prior
to use, it shall be done in a manner that prevents the raw materials and other ingredients from becoming adulterated within
the meaning of the Act.
Each lot of raw materials, product containers, and closures should be sampled, tested, or examined, as appropriate, and re-
leased for use by the quality control unit. Based upon adequate process verification, in-process controls and statistical confi-
dence, a skip-lot testing plan is an alternative to fully testing every batch provided that at least one identity test is conducted.
An appropriate amount of each lot of raw materials should be reserved for 3 years beyond the shelf life appearing on the label
of finished dietary supplements in which the raw materials were used. If adverse event reports are received (see the subsection
Adverse Event Reports), the reserved raw materials should be kept for 6 years (serious events) or 3 years (nonserious events)
from the date the first report is received.
Representative samples should be collected for testing or examination. Sampling of botanicals should be in compliance with
the provisions set in Articles of Botanical Origin á561ñ. The number of containers sampled, and the amount of material taken
from each container, should be based upon appropriate criteria such as statistical criteria for raw material variability, confi-
dence levels, and degree of precision desired, the past quality history of the supplier, and the quantity needed for analysis and
reserve where required. The following procedures should be used to collect the samples:
— The containers of raw materials selected should be cleaned, where necessary, by adequate means.
— The containers should be opened, sampled, and resealed in a manner designed to prevent contamination of their con-
tents and contamination of other raw materials, product containers, or closures.
— These containers should be identified so that the following information can be determined: name of the material sam-
pled, the lot number, the container from which the sample was taken, the date on which the sample was taken, and the
name of the person who collected the sample.
Use the following procedure to examine and test the samples:
— At least one test should be conducted to verify the identity of each raw material of a product even in cases where skip-lot
testing is used. Such tests may include any appropriate test with established sufficient specificity to determine identity,
including chemical and laboratory tests, gross organoleptic analysis, microscopic identification, or analysis of constituent
markers.
— Each raw material should be tested for conformity with all appropriate written specifications for purity, strength, and qual-
ity. However, a report of analysis may be accepted from the supplier of a raw material, provided that the manufacturer
establishes the reliability of the supplier's analyses and provided that at least one identity test is conducted on such raw
material by the manufacturer.
— Containers and closures should be tested for conformance with all appropriate written procedures. However, a certificate
of testing may be accepted from the supplier, provided that at least a visual identification is conducted on such containers
or closures by the manufacturer.
— Each lot of a raw material, rework, product container, or closure that is liable to contamination with filth, insect infesta-
tion, or other extraneous adulterant should be examined against established specifications for such contamination and
shall comply with any applicable Food and Drug Administration regulations and guidelines. Skip-lot examination should
not apply in such cases.
— Each lot of a raw material that is liable to microbiological contamination that is objectionable in view of its intended use
shall be subjected to microbiological tests before use. Raw materials either shall not contain levels of microorganisms that
may produce food poisoning or other disease in humans, or shall be otherwise treated during manufacturing operations
so that they no longer contain levels that would cause the product to be adulterated within the meaning of the Act. In
lieu of such testing by the manufacturer, a guarantee or certification of analysis may be accepted from the supplier of a
component provided that the manufacturer establishes the reliability of the supplier's analysis.
— Raw materials and other ingredients susceptible to adulteration with aflatoxin, other natural toxins, pesticides, or heavy
metals shall comply with current Food and Drug Administration regulations, guidelines, and action levels for poisonous or
deleterious substances and the requirements in Articles of Botanical Origin á561ñ, or in each monograph, before these ma-
terials or ingredients are incorporated into a finished dietary ingredient or dietary supplement. Compliance with this re-
quirement may be accomplished by analyzing these materials and ingredients for aflatoxins and other natural toxins; or,
in lieu of such testing by the manufacturer, a guarantee or certification of analysis may be accepted from the supplier of a
raw material provided that the manufacturer establishes the reliability of the supplier's analysis.
— Any lot of raw material, product container, or closure that meets the appropriate written specifications of identity,
strength, quality, and purity and related tests may be approved and released for use. Any lot of such material that does
not meet such specifications should be rejected.
Raw materials, product containers, and closures approved for use should be rotated so that the oldest approved stock is
used first. Deviation from the requirement is permitted if such deviation is temporary and adequate.
Raw materials, product containers, and closures should be retested or reexamined, as appropriate, for identity, strength,
quality, and purity and approved or rejected by the quality control unit after a specified time in storage or as necessary, e.g.,
after exposure to air, heat, or other conditions that might adversely affect the raw material, product container, or closure or
after storage of active and inactive ingredients and in-process materials for long periods of time.
Rejected raw materials, product containers, and closures should be identified and controlled under a quarantine system that
prevents their use in manufacturing or processing operations for which they are unsuitable.
Written Procedures
Written procedures should be provided for production and process controls designed to ensure that the dietary ingredients
and dietary supplements have the identity, strength, quality, and purity they are represented to possess. These procedures
should be drafted, reviewed, and approved by the appropriate organizational units and reviewed and approved by the quality
control unit. These production and process control procedures should be followed in the execution of the various production
and process control functions and should be documented at the time of performance. Any deviation from the written proce-
dures should be recorded and justified.
(1) All operations in the receiving, inspecting, transporting, segregating, preparing, manufacturing, packaging, and storing of
dietary ingredients and dietary supplements shall be conducted in accordance with adequate sanitation principles.
(2) All reasonable precautions shall be taken to ensure that production procedures do not contribute adulteration from any
source. Chemical, microbial, or extraneous-material testing procedures shall be used where necessary to identify sanita-
tion failures or possible product adulteration.
(3) All product that has become contaminated to the extent that it is adulterated within the meaning of the Act shall be re-
jected, or if permissible, treated or processed to eliminate the contamination.
(4) All product manufacturing, including packaging and storage, shall be conducted under such conditions and controls as
are necessary to minimize the potential for the growth of microorganisms, or for the adulteration of raw materials, in-
process materials, and finished product.
(5) Measures taken to destroy microorganisms, reduce the microbial load, or prevent the growth of undesirable microorgan-
isms, particularly those of public health significance, shall be adequate under the conditions of manufacture, handling,
and distribution to prevent dietary supplements and ingredients from being adulterated within the meaning of the Act.
These measures shall also comply with current regulations affecting dietary supplement products and ingredients.
(6) Work-in-process shall be handled in a manner that protects against adulteration.
(7) In-process material must be held under appropriate conditions of temperature, humidity, and light.
(8) Effective measures shall be taken to protect finished dietary ingredients and dietary supplements from adulteration by raw
materials, in-process materials, or refuse. When raw materials, in-process materials or refuse are unprotected, they shall
not be handled simultaneously in a receiving, loading, or shipping area if that handling could result in adulterated dietary
ingredients and dietary supplements. Dietary ingredients and dietary supplements transported by conveyor shall be pro-
tected against adulteration as necessary.
(9) Effective measures shall be taken as necessary to protect against the inclusion of metal or other extraneous material in
product. Compliance with this requirement may be accomplished by using sieves, traps, magnets, electronic metal detec-
tors, or other suitable effective means.
(10) Mechanical manufacturing steps such as cutting, sorting, inspecting, shredding, drying, grinding, blending, and sifting
shall be performed so as to protect dietary ingredients and dietary supplements against adulteration. Compliance with
this requirement may be accomplished by providing adequate physical protection of dietary ingredients and dietary sup-
plements from contact with adulterants. Protection may be provided by adequate cleaning and sanitizing of all processing
equipment between each manufacturing step.
(11) Heat blanching, when required in the preparation of a dietary ingredient or a dietary supplement, should be effected by
heating the product to the required temperature, holding it at this temperature for the required time, and then either
rapidly cooling the material or passing it to subsequent manufacturing without delay. Thermophilic growth and contami-
nation in blanchers should be minimized by the use of adequate operating temperatures and by periodic cleaning. Where
the blanched product is washed before filling, potable water shall be used.
(12) Intermediate of dehydrated dietary ingredients and dietary supplements that rely on the control of water (aW) for prevent-
ing the growth of undesirable microorganisms shall be processed to and maintained at a safe moisture level. Compliance
with this requirement may be accomplished by any effective means, including employment of one or more of the follow-
ing practices:
(i) Monitoring the water activity (aW) of the material;
(ii) Controlling the soluble solids–water ratio in finished product; and
(iii) Protecting finished product from moisture pickup, by use of a moisture barrier or by other means, so that the water
activity (aW) of the product does not increase to an unsafe level.
(13) Dietary ingredients and dietary supplements that rely principally on the control of pH for preventing the growth of unde-
sirable microorganisms shall be monitored and maintained at an appropriate pH. Compliance with this requirement may
be accomplished by any effective means, including employment of one or more of the following practices:
(i) Monitoring the pH and water activity, if appropriate, of raw materials, in-process material, and finished product; and
(ii) Controlling the amount of acid added to the product.
(14) When ice is used in contact with dietary ingredients and dietary supplements, it shall be made from potable water, and
shall be used only if it has been manufactured in accordance with current good manufacturing practice in manufacturing,
packing, or holding human food as outlined in 21 CFR Part 110.
Written production and control procedures should include the following, which are designed to ensure that the dietary sup-
plements have the identity, strength, quality, and purity they are represented to possess:
— The batch should be formulated with the intent to provide not less than 100 percent of the labeled or established amount
of dietary ingredient.
— Raw materials for product manufacturing should be weighed, measured, or subdivided as appropriate and the appropri-
ate signatures recorded in the batch record.
— Actual yields and percentages of theoretical yield should be determined at appropriate phases of processing.
Material scheduled for rework shall be identified as such.
Official text. Reprinted from First Supplement to USP38-NF33.
836 á2750ñ Manufacturing Practices / Dietary Supplements DSC
Equipment Identification
All compounding and storage containers, processing lines, and major equipment used during the production of a batch of a
product should be properly identified to indicate their contents and, when necessary, the phase of processing of the batch.
Sampling and Testing of In-Process Materials, Dietary Ingredients, and Dietary Supplements
To ensure batch uniformity and integrity of dietary supplements, written procedures should be established and followed that
describe the in-process controls and tests or examinations to be conducted on appropriate samples of in-process materials.
Based upon process verification, in-process controls, and statistical confidence, a skip-lot testing plan is an alternative to testing
every batch of finished products provided that at least one representative measure is performed. Control procedures should be
established to monitor the output of those manufacturing processes that may be responsible for causing variability in the char-
acteristics of in-process material and the finished product. Such control procedures may include, but are not limited to, the
following, where appropriate:
— Friability
— Weight variation
— Disintegration time
— Dissolution time
— Clarity, completeness, or pH of solutions
— Blend uniformity
In-process specifications for such characteristics should be consistent with finished product specifications. Examination and
testing of samples should ensure that the in-process material and dietary supplement conform to the established specifications.
In-process materials should be tested for identity, strength, quality, and purity as adequate, and approved or rejected by the
quality control unit during the production process, e.g., at commencement or completion of significant phases or after storage
for long periods.
Rejected or adulterated in-process materials should be identified and controlled under a quarantine system designed to pre-
vent their use in manufacturing or processing operations for which they are unsuitable and to prevent the adulteration of oth-
er products.
Written procedures should be provided describing in sufficient detail the receipt, identification, storage, handling, sampling,
examination, testing of labeling and packaging materials, or products received for packaging or labeling. Each immediate con-
tainer or grouping of immediate containers in a shipment of product received for packaging or labeling, or of packaging and
labeling materials, must be visually examined for appropriate content label, container damage, or broken seals to determine
whether the container condition may have resulted in contamination or deterioration of the received product. The supplier's
invoice, guarantee, or certification in a shipment of the received product must be visually examined to ensure that the received
product is consistent with the purchase order. Labeling and packaging materials or products received for packaging or labeling
should be quarantined until:
— Representative samples of each unique shipment, and of each unique lot within each unique shipment, of received prod-
uct for packaging or labeling, or of packaging and labeling materials, are collected;
— The quality control unit reviews and approves the documentation to determine whether the received product for packag-
ing or labeling, or packaging and labeling materials, meets the specifications; and
— The quality control unit approves the received product for packaging or labeling, or packaging and labeling materials,
and releases for use from quarantine.
Those that do not meet such specifications should be identified and rejected to prevent their use in operations for which
they are unsuitable.
A record should be kept of each shipment received of each different labeling and packaging material, or each different re-
ceived product for packaging or labeling, indicating receipt, date of examination or testing, and whether accepted or rejected.
Each unique lot within each unique shipment of received product for packaging or labeling, or of packaging and labeling
materials, must be identified in a manner that allows the recipient to trace the lot to the supplier, the date received, the name
of the received product, the status of the received product (e.g., quarantined, approved, or rejected), and to the product that
was packaged or labeled and distributed.
This unique identifier must be used whenever the disposition of each unique lot within each unique shipment of the re-
ceived product for packaging or labeling, or of packaging and labeling materials, is recorded.
Labels and other labeling materials for each different product, strength, product type, or quantity of contents should be
stored separately under conditions that will protect against contamination and deterioration and avoid mixups. Only author-
ized personnel should have access to the storage area.
Packaging and labels must be held under appropriate conditions so that the packaging and labels are not adversely affected
(e.g. contamination, deterioration).
Gang printing of labeling to be used for different products or different strengths of the same product (or labeling of the
same size and identical or similar format or color schemes) should be minimized. If gang printing is employed, packaging and
labeling operations should provide for special control procedures, taking into consideration sheet layout, stacking, cutting, and
handling during and after printing.
Printing devices on, or associated with, manufacturing lines used to imprint labeling upon the product unit label or case
should be monitored to ensure that all imprinting conforms to the print specified in the batch production record.
Obsolete and outdated labels, labeling, other packaging materials, and products received for packaging or labeling should
be destroyed and documented.
Labeling Issuance
Strict control should be exercised over labeling issued for use in product labeling operations. The control procedures em-
ployed should be in writing with sufficient detail.
Labeling materials issued for a batch should be carefully examined for identity and conformity to the labeling specified in
the master and batch production records.
Procedures should be used to reconcile the quantities of labeling issued, used, and returned, and should require evaluation
of discrepancies found. If discrepancies are found between the quantity of product finished and the quantity of labeling issued
and are outside preset limits based on historical operating data, such discrepancies should be investigated.
Returned labeling should be maintained and sorted in a manner to prevent mixups and provide proper identification.
All excess labeling bearing lot or control numbers should be destroyed and documented.
Operations
Written procedures designed to ensure that correct labels, labeling, and packaging materials are used for dietary supple-
ments should incorporate the following features:
— Prevention of mixups and cross-contamination by physical or spatial separation from operations on other products;
— Identification of the product with a lot or control number;
— Examination of packaging and labeling materials for suitability and correctness before packaging operations, and docu-
mentation of such examination in the batch production record; and
— Inspection of the packaging and labeling facilities immediately before use to ensure that all products have been removed
from previous operations. Inspection should also be made to ensure that packaging and labeling materials not suitable for
subsequent operations have been removed. Results of the inspection should be documented in the batch production re-
cords.
— Dietary ingredients and dietary supplements may be repackaged or relabeled only after the quality control unit has ap-
proved such repackaging or relabeling.
— A representative sample of each batch of repackaged or relabeled dietary ingredients and dietary supplements must be
examined to determine whether the repackaged or relabeled dietary ingredients and dietary supplements meet all estab-
lished specifications.
— The quality control unit must approve or reject each batch of repackaged or relabeled dietary ingredients and dietary sup-
plements before its release for distribution.
Tamper-Resistant Packaging
REQUIREMENTS
Each manufacturer and packer who packages a dietary supplement for retail sale shall package the product in a tamper-
resistant package, if this product is accessible to the public while held for sale. A tamper-resistant package is one having an
indicator or barrier to entry which, if breached or missing, can reasonably be expected to provide visible evidence to consum-
ers that tampering has occurred. To reduce the likelihood of substitution of a tamper-resistant feature after tampering, the in-
dicator or barrier to entry is required to be distinctive by design or by the use of an identifying characteristic (e.g., a pattern,
name, registered trademark, logo, or picture). For purposes of this section, the term “distinctive by design” means that the
packaging cannot be duplicated with commonly available materials or through commonly available processes. A tamper-resist-
ant package may involve an immediate-container and closure system, or secondary-container or carton system, or any combi-
nation of systems intended to provide a visual indication of package integrity. The tamper-resistant feature should be designed
to remain intact when handled in a reasonable manner during manufacture, distribution, and retail display.
LABELING
Each retail package of a dietary supplement covered by this section shall bear a statement that is prominently placed so that
consumers are alerted to the specific tamper-resistant feature of the package. The labeling statement should be so placed that
it will be unaffected if the tamper-resistant feature of the packaging is breached or missing. If the tamper-resistant feature
chosen to meet the requirement above is one that uses an identifying characteristic, that characteristic should be referred to in
the labeling statement. For example, the labeling statement on a bottle with a shrink band could say “For your protection, this
bottle has an imprinted seal around the neck.”
Packaged and labeled products should be examined during finishing operations to ensure that containers and packages in
the lot have the correct label. A representative sample of units should be collected at the completion of finishing operations
and visually examined for correct labeling. Results of these examinations should be recorded in the batch production or con-
trol records.
Contact Information
The manufacturer, packer, or distributor of dietary supplements is required to comply with the current labeling requirements
in the law that also include a domestic address or phone number through which an adverse event report for a dietary supple-
ment may be received.
Shelf Life
Dietary supplements should bear a date indicative of their shelf life, determined by appropriate testing, to ensure that they
meet applicable standards of identity, strength, quality, and purity at or before the labeled shelf-life date.
Shelf life should be related to any storage conditions stated on the labeling.
Warehousing Procedures
Storage and transportation of finished product shall be under conditions that will protect product against physical, chemical,
and microbial adulteration as well as against deterioration of the product and the container.
Written procedures describing the warehousing of dietary supplements should be established and followed and should in-
clude the following:
— Quarantine of finished products before disposition by the quality control unit; and
— Storage of finished products under appropriate conditions of temperature, humidity, and light so that the identity,
strength, quality, and purity of the products are not affected.
Distribution Procedures
Written procedures describing the distribution of dietary supplements shall be established and followed and should include
the following:
— A procedure whereby the oldest approved stock of a product is distributed first. (Deviation from this requirement is per-
mitted if such deviation is temporary and adequate.)
— A system by which the distribution of each lot of product can be readily determined to facilitate its recall if necessary.
The establishment of any specifications, standards, sampling plans, test procedures, or other laboratory control mechanisms
required by this chapter, including any change in such specifications, standards, sampling plans, test procedures, or other lab-
oratory control mechanisms, shall be drafted by the appropriate organizational unit and reviewed and approved by the quality
control unit. The requirements in this section should be followed and documented at the time of performance. Any deviation
from the written specifications, standards, sampling plans, test procedures, or other laboratory control mechanisms shall be
recorded and justified.
Quality control operations include the establishment of scientifically sound and appropriate specifications, standards, sam-
pling plans, and test procedures designed to ensure that raw materials, product containers, closures, in-process materials, la-
beling, products received for labeling and packaging operations as dietary supplements, and finished products conform to ad-
equate standards of identity, strength, quality, and purity. These controls include the following:
Official text. Reprinted from First Supplement to USP38-NF33.
DSC Dietary Supplements / á2750ñ Manufacturing Practices 839
— Determination of conformance to appropriate written specifications for the acceptance of each lot within each shipment
of raw materials, product containers, closures, and labeling used in the manufacture of dietary ingredients and dietary
supplements, and of products received for labeling and packaging operations as dietary supplements. (The specifications
include a description of the sampling and testing procedures used. Samples should be representative and adequately
identified. Such procedures also require appropriate retesting of any raw material, product container, or closure that is
subject to deterioration.) Based upon adequate process verification, in-process controls, and statistical confidence, a skip-
lot testing plan is an alternative to testing every batch, excluding raw materials, which require 100% identity testing.
— Determination of conformance to written specifications and a description of sampling and testing procedures for in-proc-
ess materials. (Such samples should be representative and properly identified.)
— Determination of conformance to written descriptions of sampling procedures and appropriate specifications for finished
products. (Such samples should be representative and properly identified.)
— The calibration of instruments, at suitable intervals, in accordance with an established written program containing specific
directions, schedules, limits for accuracy and precision, and provisions for remedial action in the event that accuracy
and/or precision limits are not met. Instruments not meeting established specifications shall not be used until repaired.
There should be appropriate laboratory determination of satisfactory conformance to specifications for the finished product,
including the identity and strength prior to release. Based upon adequate process verification, in-process controls, or statistical
confidence, a skip-lot or composite testing plan is an alternative to testing every batch.
There should be appropriate laboratory testing, as necessary, of each batch of dietary supplement required to be free of ob-
jectionable microorganisms. The accuracy, linearity, sensitivity, specificity, and precision of test methods employed by the firm,
when they differ from compendial methods, should be established and documented.
Written procedures should describe any sampling and testing plans, which should include the method of sampling and the
number of units per batch to be tested.
Products failing to meet established standards or specifications and any other relevant quality control criteria should be re-
jected. Rejected or adulterated dietary ingredients and dietary supplements shall be identified, stored, and disposed of in a
manner that protects against the adulteration of the other products. Reprocessing may be performed. Prior to acceptance and
use, reprocessed material must meet established standards, specifications, and any other relevant criteria. Written procedures
shall be established and followed prescribing the method for reprocessing batches or operations start-up materials that do not
conform to finished goods standards or specifications. Finished goods manufactured using such materials shall meet all estab-
lished purity, composition, and quality standards.
Stability Testing
There should be a written protocol designed to assess the stability characteristics of dietary supplements. The results of such
testing should be used in determining appropriate storage conditions and shelf life. This protocol should include the following:
— Sample size and test intervals based on statistical criteria for each attribute should be examined to ensure valid estimates
of stability;
— Storage conditions for samples retained for testing;
— Reliable, meaningful, and specific test methods should be used; and
— The dietary supplement should be tested in the same type of container–closure system as that in which the dietary sup-
plement is marketed.
An adequate number of batches of each dietary supplement should be tested to determine an adequate shelf life, and a
record of these data should be maintained. Accelerated studies combined with basic stability information on the raw materials,
dietary supplements, and container-closure systems may be used to support tentative shelf life if full shelf-life studies are not
available. Simplified stability testing procedures may be used where data from similar product formulations are available to
support a shelf-life estimation of a new product. Where data from accelerated studies are used to project a tentative shelf life
date that is beyond a date supported by actual shelf-life studies, stability studies should be conducted, including dietary sup-
plement testing at appropriate intervals, until the tentative shelf life is verified or the adequate shelf life is determined.
Reserve Samples
An appropriately identified reserve sample that is representative of each lot or batch of dietary supplement should be re-
tained and stored under conditions consistent with product labeling until at least 3 years after the shelf life of the product. The
reserve sample should be stored in the same immediate container-closure system in which the finished product is marketed or
in one that has essentially the same characteristics. The reserve sample consists of at least twice the quantity necessary to per-
form all the required tests. If an adverse event report is received, the reserve samples of dietary supplements and dietary ingre-
dients from the same lot or batch must be analyzed by an appropriate procedure to confirm their identity and determine any
adulteration or contamination. The recovered samples associated with adverse event reports from consumers, distributors, or
both should also be analyzed, following the same method used for the reserved samples, if available. The results should be
reported with other required information to the federal authority, using the required form. The reserve samples from a particu-
lar lot or batch associated with an adverse event report should be held for 6 years (serious events) or 3 years (nonserious
events) from the date when the first adverse event report is received by the manufacturer, packer, or distributor.
Any record for production, control, quality control operations, or distribution that is required to be maintained and is specifi-
cally associated with a batch of a product should be retained for at least 3 years after the shelf life of the batch.
Records should be maintained for all raw materials, product containers, closures, and labeling for at least 3 years after the
shelf life of the last lot of product incorporating the raw material or using the container, closure, or labeling.
To ensure uniformity from batch to batch, master production and control records for each product should be prepared, dat-
ed, and signed by one person and independently checked, dated, and signed by a second person from the quality control
unit.
Master production and control records should include the following:
— The name and strength of the product;
— The name and weight or measure of each dietary ingredient per unit or portion or per unit of weight or measure of the
product, and a statement of the total weight or measure of any dosage unit;
— A complete list of raw materials designated by names or codes sufficiently specific to indicate any special quality charac-
teristic;
— An accurate statement of the weight or measure of each raw material, using the same weight system (metric, avoirdupois,
or apothecary) for each raw material;
— A statement concerning any calculated excess of raw material;
— A statement of theoretical weight or measure at appropriate phases of processing;
— A statement of theoretical yield, including the maximum and minimum percentages of theoretical yield beyond which
investigation is required;
— A description of the product containers, closures, and packaging materials, including a specimen or copy of each label
and all other labeling signed and dated by the person or persons responsible for approval of such labeling or, in lieu of
specimens or copies of each label or other labeling, a positive identification of all labeling used; and
— Complete manufacturing and control instructions, testing procedures, acceptance limits, special notations, and precau-
tions to be followed.
— Specific actions necessary in order to perform and verify points, steps, or stages in the manufacturing process where con-
trol is necessary to ensure the quality of dietary ingredients and dietary supplements, and to ensure that dietary ingredi-
ents and dietary supplements are packaged and labeled as specified in the master production record.
(a) Such specific actions must include verifying the weight or measure of any component and verifying the addition of
any component; and
(b) For manual operations, such specific actions must include:
(i) One person weighing or measuring a component and another person verifying the weight or measure; and
(ii) One person adding the component and another person verifying the addition.
— Corrective action plans for use when a specification is not met.
Batch production and control records should be prepared for each batch of product produced and should include complete
information relating to the production and control of each batch. These records should be reviewed and signed by a second
person from the quality control unit. These records should include accurate reproduction of the appropriate master production
or control record and documentation that each significant step in the manufacture, processing, packing, or holding of the
batch was accomplished, including the following:
— Dates;
— Identity of individual major equipment and lines used;
— Specific identification of each batch of raw material or in-process material used;
— Weights and measures of raw materials used in the course of processing;
— In-process and laboratory control results;
— Inspection of the packaging and labeling areas before and after use;
— A statement of the actual yield and a statement of the percentage of theoretical yield at appropriate phases of processing;
— Description of product containers and closures used;
— Complete labeling control records, including:
(a) The unique identifier assigned to packaging and labels used, the quantity of the packaging and labels used, and,
when label reconciliation is required, reconciliation of any discrepancies between issuance and use of labels; and
(b) An actual or representative label, or a cross-reference to the physical location of the actual or representative label
specified in the master manufacturing record;
— Any sampling performed;
— Identification of the persons performing and directly supervising or checking any step in the operation;
— Any investigation made;
— The results of any tests or examinations conducted on packaged and labeled dietary supplements (including repackaged
or relabeled dietary supplements), or a cross-reference to the physical location of such results;
— Documentation at the time of performance that quality control unit:
(a) Reviewed the batch production record, including:
(i) Review of any required monitoring operation, and
(ii) Review of the results of any tests and examinations, including tests and examinations conducted on compo-
nents, in-process materials, finished batches of dietary supplements, and packaged and labeled dietary ingredi-
ents and dietary supplements;
(b) Approved or rejected any reprocessing or repackaging;
(c) Approved and released, or rejected, the batch for distribution, including any reprocessed batch; and
(d) Approved and released, or rejected, the packaged and labeled dietary supplement, including any repackaged or rela-
beled dietary supplement;
— Documentation at the time of performance of any required material review and disposition decision; and
— Documentation at the time of performance of any reprocessing.
Records for Raw Materials, Packaging, and Labels and for Product Received for Packaging or
Labeling as a Dietary Supplement
Laboratory Records
Laboratory records should include complete data derived from all tests necessary to ensure compliance with established
specifications and standards, including examinations and assays, as follows:
— A description of the sample received for testing with identification of source (that is, location from where sample was ob-
tained), quantity, lot number or other distinctive code, and date sample was taken.
— A statement of each method used in the testing of the sample.
— A statement of the weight or measure of sample used for each test, where appropriate.
— A complete record of all data secured in the course of each test, including all graphs, charts, and spectra from laboratory
instrumentation, properly identified to show the specific raw material, product container, closure, in-process material, or
finished product, and lot tested.
— A record of all calculations performed in connection with the test, including units of measure, conversion factors, and
equivalency factors.
— A statement of the results of tests and how the results compare with established standards of identity, strength, quality,
and purity for the raw material, product container, closure, in-process material, or finished product tested.
— The initials or signature of the person who performs each test and the date(s) the tests were performed.
Complete records should be maintained of any modification of an established method employed in testing. Such records
should include the reason for the modification and data to verify that the modification produced results that are at least as
accurate and reliable for the material being tested as the established method.
Official text. Reprinted from First Supplement to USP38-NF33.
842 á2750ñ Manufacturing Practices / Dietary Supplements DSC
Complete records should be maintained of any testing and standardization of laboratory reference standards, reagents, and
standard solutions, the periodic calibration of laboratory instruments, and all stability testing performed. Any deviation should
be reviewed and signed by the management of the quality control unit.
Distribution Records
Distribution records should contain the name and strength of the product, name and address of the consignee, date and
quantity shipped, and lot or control number of the finished product.
Record Keeping
The manufacturer, packer, or distributor of dietary supplements must keep all required records, as shown in this chapter, for
3 years beyond the shelf life of dietary supplements associated with those records. If adverse event reports are received, those
records must be kept for additional 6 years from the date when the first report is received. All records must be accessible by
the regulatory authority when requested.
Records must be kept as original records, as true copies (such as photocopies, microfilm, microfiche, or other accurate repro-
ductions of the original records), or as electronic records.
All electronic records must comply with part 11 of Code of Federal Regulations Title 21 (21 CFR part 11).
If reduction techniques are used, such as microfilming, suitable reader and photocopying equipment must be readily availa-
ble to auditors and inspectors.
Complaint Files
Written procedures describing the handling of all written and oral complaints regarding a dietary supplement shall be estab-
lished and followed. These procedures should include provisions for review by the quality control unit of any complaint involv-
ing the possible failure of a product to meet any of its specifications and a determination as to the need for an investigation.
Each complaint should be recorded in a file designed especially for dietary supplement complaints. Written records should
be maintained until at least 3 years after the shelf life of the product, or 3 years after the date when the complaint was re-
ceived, whichever is longer.
The written record should include the following information, where known: the name and strength of the product, lot num-
ber, name of complainant, nature of complaint, and reply to complainant.
If an investigation is necessary, the written record should include the findings of the investigation and follow-up.
The review and investigation of the product complaint by a qualified person, the review by quality control unit about
whether to investigate a product complaint, and the findings and follow-up action of any investigation performed must ex-
tend to all relevant batches and records.
Adverse event reports include reports on any health-related adverse event associated with the use of a dietary supplement
that is adverse. It includes both nonserious and serious adverse event reports.
The manufacturer, packer, or distributor of a dietary supplement (called the responsible person) whose name appears on the
label shall be responsible for keeping reports of all nonserious adverse events along with any related records (e.g., records of
communications with the person who reported the nonserious event). All such records of nonserious adverse events should be
kept for 6 years.
The responsible person whose name appears on the label shall also be responsible for reporting any serious adverse event
reported to it, and associated with a dietary supplement that is marketed and used in the same country, to the regulatory
authority as soon as appropriate, but no later than 15 business days after receipt of the report, using the appropriate form as
defined by the regulation (http://www.fda.gov/Food/DietarySupplements/Alerts/ucm111110.htm). A serious adverse event is
an event that results in any of following:
1. Death,
2. A life-threatening experience,
3. Inpatient hospitalization,
4. A persistent or significant disability or inability,
5. A congenital anomaly or birth defect, or
6. A condition that requires, according to reasonable medical judgment, a medical or surgical intervention to prevent one of
the five outcomes listed above.
A retailer whose name appears on the label as a distributor may, by agreement, authorize the manufacturer or packer to
submit the required reports to the regulatory authority, as long as the retailer directs all received adverse event reports to the
manufacturer or packer. Each serious adverse event report should include a copy of the product’s label, the information descri-
bed in the preceding section Complaint Files, and if possible, the contact information of the complainant; daily intake; alcohol
consumption and amount; use of prescription medicine and OTC medicine, including a daily dose; and other medical informa-
tion. The information associated with personal identification and medical records should be obtained only for the reports and
kept safe from disclosure. Any new medical information that is related to an already submitted serious adverse event report
that is received within 1 year of the initial report shall be submitted to the regulatory authority as soon as appropriate, but no
later than 15 business days after receipt of the information. The records related to each report of a serious adverse event re-
ceived by the manufacturer, packer, or retailer should be maintained for 6 years. The authorized person who is designated by
the regulatory authority should be permitted access to those records.
Returned products should be identified as such and held. If the conditions under which returned dietary ingredients and
dietary supplements have been held, stored, or shipped before or during their return, or if the condition of the product, its
container, carton, or labeling, as a result of storage or shipping, casts doubt on the safety, identity, strength, quality, or purity
of the product, the returned product should be destroyed unless examination, testing, or other investigations prove the prod-
uct meets appropriate standards of safety, identity, strength, quality, or purity. The returned products associated with adverse
events must be destroyed after a sufficient sample of products is stored for the purpose of further investigation only. The prod-
ucts related to the adverse event that have been returned should be kept for 6 years (serious events) or 3 years (nonserious
events) from the date when the first report is received. A product may be reprocessed provided that the subsequent product
meets adequate standards, specifications, and characteristics. Records of returned products should be maintained and should
include the name and label potency of the product, lot number (or control number or batch number), reason for the return,
quantity returned, date of disposition, and ultimate disposition of the returned product. If the reason for a product being re-
turned implicates associated batches, an appropriate investigation is necessary.
Products that have been involved in adverse events or subjected to improper storage conditions, including extremes in tem-
perature or humidity, smoke, fumes, pressure, age, or radiation due to natural disasters, fires, accidents, or equipment failures
should not be salvaged and returned to the marketplace. Whenever there is a question whether products have been subjected
to such conditions, salvaging operations may be conducted only if there is (a) evidence from laboratory tests and assays that
the products meet all applicable standards of identity, strength, quality, and purity, and (b) evidence that the products and
their associated packaging were not subjected to improper storage conditions as a result of the disaster or accident. Organo-
leptic examinations should be accepted only as supplemental evidence that the dietary supplement meets appropriate stand-
ards of identity, strength, quality, and purity. Records including name, lot number, and disposition should be maintained for
salvaged products. If the products are involved in adverse events, the instructions described in the preceding section Records
and Reports should be followed.
Some dietary ingredients and dietary supplements, even when produced under current good manufacturing practice, con-
tain natural or unavoidable defects that at low levels are not hazardous to health. The Food and Drug Administration estab-
lishes maximum levels for these defects in dietary ingredients and dietary supplements produced under current good manufac-
turing practice and uses these levels in deciding whether to recommend regulatory action.
Defect action levels are established for dietary ingredients and dietary supplements whenever it is necessary and feasible to
do so. These levels are subject to change upon the development of new technology or the availability of new information.
Compliance with defect action levels does not excuse violation of the requirement in section 402(a)(4) of the Act that diet-
ary ingredients and dietary supplements shall not be prepared, packed, or held under unsanitary conditions or the require-
ments in this part that manufacturers, distributors, and holders of both dietary ingredients and dietary supplements shall ob-
serve current good manufacturing practice. Evidence indicating that such a violation exists causes a dietary ingredient and a
dietary supplement to be adulterated within the meaning of the Act, even though the amounts of natural or unavoidable de-
fects are lower than the currently established defect action levels. The manufacturer, distributor, and holder of a dietary ingre-
dient or a dietary supplement shall at all times utilize quality control operations that reduce natural or unavoidable defects to
the lowest level currently feasible.
The mixing of a dietary ingredient or dietary supplement containing defects above the current defect action level with an-
other lot of dietary ingredient or dietary supplement is not permitted and renders the final product adulterated within the
meaning of the Act, regardless of the defect level of the final product.
A compilation of the current defect action levels for natural or unavoidable defects in dietary ingredients and dietary supple-
ments that present no health hazard may be obtained upon request from the Industry Activities Staff (HFS-565), Center for
Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD
20740-3835.
GLOSSARY
Act means Federal Food, Drug and Cosmetic Act (United States Code [U.S.C.] Title 21, Chapter 9).
Acceptance criteria are the product specifications and acceptance or rejection criteria, such as acceptable quality level and
unacceptable quality level, with an associated sampling plan, that are necessary for making a decision to accept or reject a lot
or batch (or any other convenient subgroups of manufactured units).
Adequate means that which is needed to accomplish the intended purpose in keeping with good public health practice.
Adverse event means any health-related event that is adverse and that is associated with the use of a dietary supplement.
Adverse event report means a report of an adverse event (see definition above). (See also Serious adverse event report.)
Batch is a specific quantity of a finished product or other material that is intended to have uniform character and quality,
within specified limits, and is produced according to a single manufacturing order during the same cycle of manufacture.
Blanching means a prepackaging heat treatment of a dietary ingredient and a dietary supplement for a sufficient time and at
a sufficient temperature to partially or completely inactivate the naturally occurring enzymes and to effect other physical or
biochemical changes in the product.
Composition is (1) the identity of a dietary ingredient or dietary supplement, and (2) the concentration of a dietary ingredi-
ent (e.g., weight or other unit of use/weight or volume), or the potency or activity of one or more dietary ingredients, as indi-
cated by appropriate procedures.
Dietary ingredient is an ingredient intended for use or used in a dietary supplement that is
— a vitamin;
— a mineral;
— an herb or other botanical;
— an amino acid;
— a dietary substance for use by humans to supplement the diet by increasing the total dietary intake, or a concentrate,
metabolite, constituent, extract; or
— a combination of any of the foregoing ingredients.
Dietary supplement is a product (other than tobacco) that is intended to supplement the diet and that bears or contains one
or more of the following dietary ingredients: a vitamin, a mineral, an herb or other botanical, an amino acid, a dietary sub-
stance for use by humans to supplement the diet by increasing the total daily intake, or a concentrate, metabolite, constituent,
extract or combination of these ingredients, that is intended for ingestion in a pill, capsule, tablet or liquid form, that is not
represented for use as a conventional food or as the sole item of a meal or diet, and that is labeled as a dietary supplement,
and includes products such as new drug, certified antibiotic, or licensed biologic that was marketed as a dietary supplement or
food before approval, certification, or license unless a sanitary authority waives this provision.
Inactive ingredient is any raw material other than a dietary ingredient.
In-process material is any material fabricated, compounded, blended, ground, extracted, sifted, sterilized, or processed in any
other way that is produced for, and used in, the preparation of the dietary supplement.
Lot is a batch, or a specific identified portion of a batch, having uniform character and quality within specified limits.
Lot number, control number, or batch number is any distinctive combination of letters, numbers, or symbols, or any combina-
tion of them from which the complete history of the manufacture, processing, packing, holding, and distribution of a batch or
lot of finished dietary ingredient, dietary supplement, or other material can be determined.
Manufacture or manufacturing includes all operations associated with the production of dietary ingredients and dietary sup-
plements, including packaging and labeling operations, testing, and quality control of a dietary ingredient or dietary supple-
ment.
Microorganisms means yeast, molds, bacteria, and viruses and includes, but is not limited to, species having public health
significance. The term “undesirable microorganisms” includes those microorganisms that are of public health significance, that
subject a dietary ingredient or a dietary supplement to decomposition, that indicate that a dietary ingredient or dietary supple-
ment is contaminated with filth, or that otherwise may cause a dietary ingredient or a dietary supplement to be adulterated
within the meaning of the Act. Occasionally in these regulations, the adjective “microbial” is used instead of an adjectival
phrase containing the word “microorganism.”
Pest refers to any objectionable animals or insects including, but not limited to, bird, rodents, flies, and larvae.
Plant means the building or facility or parts thereof, used for or in connection with the manufacturing, packaging, labeling,
or holding of a dietary ingredient and a dietary supplement.
Process evaluation is a set of tests performed on a process intended to evaluate its capacity to consistently produce the results
that it is intended for.
Quality control operation is a planned and systematic procedure for taking all actions necessary to prevent a dietary ingredi-
ent and a dietary supplement from being adulterated.
Quality control unit is any person or organizational element designated by the firm to be responsible for the duties relating to
quality control operations.
Raw material is any ingredient intended for use in the manufacture of a dietary ingredient or dietary supplement, including
those that may not appear in such finished product. (A dietary ingredient is a raw material when considering the manufacture
of a dietary supplement.)
Representative sample is a sample that consists of a number of units that are drawn based on rational criteria such as random
sampling and is intended to ensure that the sample accurately portrays the material being sampled.
Rework is a clean, unadulterated material that has been removed from processing for reasons other than unsanitary condi-
tions or that has been successfully reconditioned by reprocessing and that is suitable for use in the manufacture of a dietary
ingredient or a dietary supplement.
Sanitizing is to adequately treat equipment, containers, or utensils by a process that is effective in destroying vegetative cells
of microorganisms of public health significance and in substantially reducing other undesirable microorganisms but without
affecting the product or its safety for the consumer.
Serious adverse event report means a report of an adverse event that is termed serious because it meets certain criteria (see
the subsection Adverse Event Reports). The Dietary Supplement and Nonprescription Drug Consumer Protection Act requires
manufacturers and distributors of dietary supplements and OTC drugs to report all serious adverse events to the Secretary of
the Food and Drug Administration (FDA). This is an entirely new requirement for dietary supplements.
Shall is used to state requirements that must be met under the provisions of this guideline.
Shelf life is the period of time after manufacturing in which the dietary supplement is ensured to meet applicable standards
of identity, strength, quality, and purity.
Shelf-life (Use by) date is the date beyond which the dietary supplement is no longer ensured to meet applicable standards of
identity, strength, quality, and purity.
Should is used to state recommended or advisory procedures or identify recommended equipment.
Skip-lot testing (or sampling) is a reduced level of testing (or sampling) for a particular specified parameter(s) based upon one
or more of the following:
— Statistical analysis of an adequate quantity of historical test data;
— Statistical confidence in the capability of the manufacturing process as determined by suitable verification; or
— Ongoing monitoring of the process using recognized statistical process control (SPC) techniques.
Strength means the concentration of the active substance (weight/weight, weight/volume, or unit of use/volume or weight
basis); and/or the potency, that is, the activity of the product as indicated by appropriate laboratory tests.
Water activity (aW) is a measure of the free moisture in a dietary ingredient or dietary supplement and is the quotient of the
water vapor pressure of the substance divided by the vapor pressure of pure water at the same temperature.