National Enforcement Investigations Center, Denver
U.S. Environmental Protection Agency
Office of Enforcement
EVALUATION OF CHEMISTRY-RELATED ASPECTS OF
FIFRA ANO TSCA HEALTH EFFECTS STUDIES FOR
GLP/DATA AUDIT INSPECTIONS
January 1987

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EVALUATION OF CHEMISTRY-RELATED ASPECTS OF
FIFRA AND TSCA HEALTH EFFECTS STUDIES FOR
GLP/DATA AUDIT INSPECTIONS
January 1987
Dean F. Hill
National Enforcement Investigations Center
Denver, Colorado

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CONTENTS
INTRODUCTION	1
I BACKGROUND AND GENERAL INFORMATION	2
II. TEST AND CONTROL SUBSTANCE RECEIPT,
STORAGE AND DISTRIBUTION	6
TEST SUBSTANCE RECEIPT	7
TEST SUBSTANCE STORAGE	7
TEST SUBSTANCE REPOSITORY	8
TEST SUBSTANCE LABELING AND GENERAL USE	8
TEST SUBSTANCE DISTRIBUTION	9
CONTROL SUBSTANCES	10
FEED STORAGE	10
CONTROL SUBSTANCES ANALYSIS	11
III DESCRIPTION, CHARACTERIZATION, ASSAY AND STABILITY OF TEST
SUBSTANCE	12
DESCRIPTION OF TEST SUBSTANCE	12
TEST SUBSTANCE ASSAY	13
TEST SUBSTANCE CHARACTERIZATION	14
TEST SUBSTANCE STABILITY	14
QUALITY ASSURANCE AND ARCHIVING	15
INSTRUMENT LOGBOOKS	16
PERSONNEL	16
GENERAL	16
IV FORMULATION OF TEST SUBSTANCE IN CARRIER	18
ORGANIZATION	18
STANDARD OPERATING PROCEDURES (SDPs)	19
DIET MIXING DIRECTION	19
TEST SUBSTANCE HANDLING	20
DIET PREPARATION	20
EQUIPMENT	21
DIET STORAGE	22
NON-FEED MIXTURES	22
WATER-BASED CARRIER	23
DIET SAMPLING FOR ANALYSIS	23
V VERIFICATION OF HOMOGENEITY AND STABILITY OF TEST SUBSTANCE
IN CARRIER	25
GENERAL	25
METHODOLOGY	26
ACCEPTANCE CRITERIA	26
ANALYTICAL QUALITY CONTROL	27
ARCHIVING AND QUALITY ASSURANCE	27
VI CONFIRMATION OF NOMINAL TEST SUBSTANCE LEVELS IN CARRIER	28
PROTOCOLS AND SOPs	29
SAMPLING	29
SAMPLE CONTROL	29
TURN-AROUND TIME AND SAMPLE STORAGE	30
METHODOLOGY	30
PERSONNEL	31
ANALYTICAL QUALITY CONTROL	31
ANALYTICAL REPORTS	32
ANALYTICAL REFERENCE STANDARDS	32
INSTRUMENTATION	34
HOUSEKEEPING	34
ANALYTICAL RAW DATA	34
QUALITY ASSURANCE	35
VII.
REFERENCES

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INTRODUCTION
This manual is primarily intended for NEIC and other chemists that
audit non-clinical health effects studies as part of EPA inspections con-
ducted in support of Good Laboratory Practices (GLP) compliance efforts.
The guidance provided herein should also prove useful for conducting GLP
inspections at chemistry laboratories furnishing analytical support to on-
going health effects studies. NEIC and other Agency chemists will often
serve as the official EPA inspector for a GLP-related inspection; however,
this manual does not provide guidance for the fulfillment of these official
duties.
These guidelines are restricted to the chemistry aspects of health
effects studies, as both TSCA and FIFRA GLP Standards regulations for these
types of studies have been promulgated. The manual will be updated or
supplemented accordingly, as FIFRA regulations for environmental effects
testing, chemical fate studies, efficacy and/or residue methodology develop-
ment are promulgated. Future revisions will also specifically address
inhalation studies, clinical chemistries, feed, water and bedding analyses
for contaminants, once more experience has been gained in these subject
areas.
The criteria and procedures contained herein are intended to supple-
ment those currently provided in other Agency directives and guidelines,
particularly those given in the EPA GLP Inspector's Manual (1). Additional
guidance documents are referenced in Chapter IX and should be consulted, as
necessary.

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I. BACKGROUND AND GENERAL INFORMATION
Under the authority of the Federal Insecticide, Fungicide and Rodenti-
cide Act (FIFRA) and the Toxic Substances Control Act (TSCA), Good Labora-
tory Practices (GLP) inspections may be conducted at in-house, contract or
academic laboratories. These inspections are conducted for the purposes of
evaluating the GLP Standards compliance and/or the overall quality, accuracy
and reliability of ongoing and completed health effects studies. For
ongoing studies that will be submitted to EPA for regulatory purposes
(generally market approval), a GLP Standards inspection will normally be
performed, whereas, for completed and reported studies, "data audits" will
be conducted.
In addition to health-effects studies, the TSCA GLP Standards regula-
tions also address environmental effects (including chemical fate) testing.
Si mi 1i ar regulations for FIFRA-related tests and studies have not yet been
promulgated. Efficacy and chemical/physical testing are not currently
covered under either Act. Thus, data audits may be conducted for a wide
variety of reported studies; however, GLP Standards inspections will be
contingent upon the applicable law (TSCA or FIFRA) and the type of study.
Generally, specialists, or otherwise knowledgeable individuals, will be
available for review of specific scientific elements of each GLP Standards
or data audit inspection. This manual is intended primarily for use by a
trained or experienced chemist. It may also prove useful to inspectors or
others involved in inspections or report reviews.
The following general criteria are common to all GLP Standards and/or
Data Audit inspections and, thus, will provide the chemist or investigator
with ideas on the broader areas to address. These criteria are outlined
here for general emphasis only. Additional details are provided in the
specific study component sections that follow.
Accuracy - Are the reported findings and conclusions supported by
the raw data? (Generally applicable to data audit inspections
only.)

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Completeness - Are all findings and conclusions in the final
report supported by retained raw data and other original records?
[missing raw data may also represent a potential pesticide regis-
tration regulation violation according to the requirements of
40 CFR Part 169.2(k)].
Documentation - Are all raw data fully identified with respect to,
by whom, when and why (if necessary) it was generated or entered?
Are all raw data entries made in pen? Are corrections made accord-
ing to GLP Standards criteria (i.e., single line-out with initials,
date and explanation)? Are all data filed in an easily accessible
manner and cross-referenced, as necessary?
Is (was) the study conducted according to the applicable GLP Stand-
ards regulations?
Are (were) the individual supporting .study components scientifT
cally sound?
Is (was) the study governed by and conducted according to a prop-
erly approved protocol?
Is (was) the study conducted in reasonable conformance with
established applicable Agency or other recognized testing guide-
lines?
Are (were) the archives and quality assurance unit (QAU) consis-
tent with GLP Standards regulations?
Was the final report complete, clearly written and consistent
with GLP Standards regulations?

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The following components of health effects studies should be reviewed
and evaluated by a professional chemist.
Test and control substance receipt, storage and distribution
(inventory)
Test and control substance labeling
Characterization, assay and stability of test substance
Formulation of test substance in carrier
Verification of homogeneity and stability of test substance in
carrier
Confirmation of nominal test substance concentrations in carrier
Analysis of feed, water and bedding, for chemical contaminants
Clinical chemistry analyses and reliability of findings
The above topics are all directly, or in some cases (i.e., bedding
analysis) indirectly, addressed in both the TSCA and FIFRA GLP Standards
regulations. Inhalation studies will be treated separately because of the
unique exposure and measurement systems involved; however, the overall GLP
Standards requirements are the same as for other studies. Clinical chem-
istry and feed, water and bedding contaminant analyses will also be addres-
sed in a future edition of this manual.
The following represent the minimal documentation that the chemist or
inspector should collect during a GLP/data audit inspection. These docu-
ments should be referenced in the report and attached as exhibits.
Curricula vitae for all senior study staff, and for junior staff,
as necessary, to document suspected problems

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Copies of current GLP-required Master Schedule(s)
Organizational chart(s) of current corporate and technical align-
ments and older organizational structures, as appropriate
Copies of routinely used laboratory forms (as examples)
Copy of the index to the SOPs, if available
Copies of all raw data, records, methods, bench sheets and inter-
nal reports which demonstrate, reflect or otherwise document ille-
gal, poor, improper or suspect practices or inconsistencies.
These should be initialed and dated by the inspector or investi-
gating chemist when received. The inspector should officially
seal records or specimens if gross violations, tampering or crimi-
nal intent is suspected.
Sampling of test substance, carrier, dosed carrier, specimens or other
non-documents should be accomplished only as a last resort. A photograph
may be adequate. If sampling is necessary to document suspected violations,
it must be performed in consultation with the inspector and full considera-
tion must be given to sample splitting, custody, identification, preserva-
tion and official receipt. Further guidance regarding sampling is provided
in the TSCA Inspection Manual (3).
All documents, samples and photographs should be listed on the appro-
priate FIFRA or TSCA Receipt for Sample. Consideration must be given TSCA
Confidential Business Information (CBI) for any TSCA-related study or GLP
Standards inspectional documents, particularly when proprietary formulae,
manufacturing processes or financial records are involved. Further guidance
regarding TSCA CBI is provided in references (3) and (6).

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II. TEST AND CONTROL SUBSTANCE RECEIPT, STORAGE AND DISTRIBUTION
Title 40 Code of Federal Regulations (CFR), parts 160.107 and 792.107
provide, respectively, the FIFRA and TSCA GLP Standards criteria for test
substance handling in the non-clinical health testing laboratory. The "test
substance" is the chemical or mixture of chemicals that is under study. It
is further defined in parts 160.3(o)(l)/(2) and 792.3(o) of the FIFRA and
TSCA GLP Standards regulations, respectively. Facility requirements for
test and control substances are given in parts 792.47 (TSCA) and 160.47
(FIFRA).
Parts 160.105(c) of FIFRA and 792.105(c) of TSCA GLP Standards regu-
lations address labeling of test and control substances, and part (d) of
these two sections addresses retention of reserve samples for each batch of
test and control substance used during studies of at least 4 weeks dura-
tion. These reserve samples are to be retained for periods of time, as
specified in the "Retention of Records" sections of each of the appropriate
GLP Standards regulations.
Characterization, identity, strength, stability and manufacture of the
test substance are addressed in parts 160.105 and 792.105 of the FIFRA and
TSCA GLP Standards regulations, respectively. Guidelines for addressing
these specific topics are provided separately in the next section of this
manual.
The "control substance" is defined in both the TSCA and FIFRA GLP Stand-
ards regulations as ". . .any chemical substance or mixture or any other
material other than a test substance that is administered to the test system
in the course of the study for the purpose of establishing a basis for com-
parison with the test substance." Thus, the control substance normally is
the undosed diet, tap water or room air, including the addition of any
excipient or vehicle that is used to assist test substance solubility or
compatability. The control substance would also encompass any positive
control material that is utilized for certain types of health effects
studies.

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During the inspection, the following questions should be answered
regarding the storage, labeling, handling, transfer and reserve sample
retention for test and control substances through (1) review of available
raw data and study records, (2) personal interviews and/or (3) laboratory
visits.
TEST SUBSTANCE RECEIPT
When and from whom was the designated batch of test substance
received? How was receipt documented in laboratory records (i.e.,
receipts, invoices, bill of lading, etc.)?
How much was received?
How was it packaged and labeled?
What was the physical description (i.e., state, color, consis-
tency and odor)?
Do these descriptive factors agree with the final report and other
literature values for the test substance, if available? (If more
than one batch of test substance was used during the study, these
descriptions are to be obtained for each.)
Is the description consistent in each study report where the same
test substance (or batch) is described as having been used? (It
may be necessary to observe the reserve sample of test substance,
if available, if there is any discrepancy or confusion regarding
its description or mixup in use.)
TEST SUBSTANCE STORAGE
How was the test substance stored prior to and during the study?

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Were sponsor's, manufacturer's or supplier's storage directions
followed?
Was the material subdivided from a larger container for more con-
venient use?
How were these smaller containers labeled so as to preclude mixup?
Was the test substance kept frozen or refrigerated when not in
use? If so, was it allowed to equilibrate to ambient temperature
in a dessicator before opening so as to minimize moisture
condensation?
TEST SUBSTANCE REPOSITORY
Does the biological testing facility maintain a formal test sub-
stance repository or is test substance distribution controlled by
the formulating or other department?
Are there approved and adequate SOPs governing receipt, storage,
labeling and distribution of test substances?
Are storage facilities adequate for storage from the standpoint
of both space and environmental controls?
TEST SUBSTANCE LABELING AND GENERAL USE
If the study is ongoing, are the test substance containers prop-
erly labeled by name, CAS number, code or batch number, expira-
tion date (if applicable) and storage requirements?
Has a reserve sample been retained for studies exceeding 4 weeks?

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Has the stability been verified for the period of use during the
study? (Also, see next section)
If stability has not been established, is (was) there an SOP to
establish stability data concurrently with the in-life portion of
the study?
How are dispensed subsamples of the test substance identified so
that mixup or contamination is precluded?
Is (was) the storage of the test substance (and associated sub-
samples) consistent with the approved protocol and/or established
literature?
TEST SUBSTANCE DISTRIBUTION
How is (was) the use of and/or dispensing of the test substance
monitored?
Is (was) a double bookkeeping system used (i.e., is weight of
removed subsamples recorded as well as the bulk container weight
differences)?
How are dispensing data kept?
Are (were) dispensing logbooks or bench sheets properly and con-
sistently signed and dated?
Does security appear to be adequate regarding the test substance
and the distribution records?

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CONTROL SUBSTANCES
What is (was) the negative control substance?
If the control substance is (was) undosed feed or chow, are (were)
SOPs available and being followed regarding receipt, storage,
usage and shelf-life disposal?
Does (did) the control substance include all vehicles, excipients
or other substances used to aid the dissolution, adhesion, compat-
ibility or stability of the test substance?
Is (was) incoming feed or diet logged as to date received and
subsequent dispensing to assure "first in - first out" use?
Is (was) the feed or other diet material analyzed and/or certified
for nutrient and physical composition?
Is (was) the nutrient composition consistent with the NTP, NIH or
other recognized criteria?
FEED STORAGE
Is the feed storage area clean and well organized?
Are feed hoppers adequately labeled?
Are feed bags stored on pallets or shelves (i.e., off the floor)?
Is there any evidence of insect or rodent infestation in the diet
storage area?
What insecticides and/or rodenticides are being used, if any, in
the diet storage and mixing areas?

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Are there odors or reports that suggest insecticide or other
pesticide usage in the feed storage area?
CONTROL SUBSTANCE ANALYSIS
Has the control substance(s) (i.e., feed, corn oil, organic sol-
vent and/or water) been periodically analyzed for chemical con-
taminants? If so:
What contaminants?
Who performed the analysis?
What methodology was utilized and what were the detection
limits?
Were (are) reports of these analyses available in the study
files?

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III. DESCRIPTION, CHARACTERIZATION, ASSAY AND STABILITY OF TEST SUBSTANCE
Test substance characterization, assay and stability testing (ref.
FIFRA - 40 CFR 160.106 and TSCA - 40 CFR 792.105) will often be conducted
by a laboratory (generally the sponsor) other than the analytical laboratory
located at the toxicological testing facility. For this reason, special
efforts are required before the inspection to assure that the necessary raw
data, reports and other records are made available at the inspection site.
If the necessary data are not available at the inspection site, the sponsor
should be requested, by letter, to provide such data. If there is any ques-
tion regarding the identity, integrity, quality or the completeness of the
data, then the performing analytical laboratory should be inspected to verify
and document findings. The reserve sample, or a portion thereof, may also
need to be provided at the audit site if not retained in the toxicological
laboratory archives. Ideally the reserve test substance sample will have
been retained in frozen storage.
The following questions should be answered satisfactorilyby examina-
tion of raw data, reports and records, employee interviews and/or visits to
appropriate laboratory areas.
DESCRIPTION OF TEST SUBSTANCE
Is (was) the test substance technical grade, analytical grade, a
formulation or something else?
What was the manufacturing or synthesis process used to produce
and refine the test substance?
Was it a composite?
If so, what were the source(s) and identification(s) of the indi-
vidual batches that went into the composite?

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What cleanup steps were used in the test substance preparation?
How does the characterization of the test substance compare with
material currently produced and/or on the market? (This informa-
tion may not always be readily available.)
TEST SUBSTANCE ASSAY
What is (was) the assay value of the active or principal
ingredient?
What laboratory performed the assay?
Who performed the assay? What were his or her qualifications?
What method was used to assay the active ingredient?
Was the method validated?
Was the method scientifically sound from the standpoint of selec-
tivity and reproducibility?
What was the source of the analytical reference standard used for
the assay?
How was the reference standard characterized and assayed?
Are the raw data for the analytical standard characterization
complete, fully identified and available for review?
Were the assay data generated under GLP Standards controlled con-
ditions?

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TEST SUBSTANCE CHARACTERIZATION
To what degree has the test substance been characterized with
respect to:
(a)	By-products
(b)	Inert additives
(c)	Contaminants
(d)	Decomposition products
(e)	Additives or inert ingredients
What laboratory performed the characterization analyses?
Who performed the characterization analyses?
What were the qualifications of the analysts?
What analytical methods were used to generate the characteriza-
tion data?
Were the methods appropriate and scientifically sound?
Were the characterization data summarized in a separate report?
Were the characterization data developed according to the GLP
Standards criteria?
TEST SUBSTANCE STABILITY
Are (were) data available to substantiate the test substance sta-
bility over the time period it was used (or will be used) in the
toxicology study?

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What methods were used for these additional assays?
How often were the stability assays performed?
How was the stability test material stored?
Were storage conditions different from those used during the
toxicology test?
Who performed the stability analyses?
What were his or her qualifications (if the analyst was different
from the individual who performed the original assay)?
Are the stability raw data complete, fully identified as to by
whom, when and how they were generated?
Do the raw data support the reported values? Have the stability
raw data been adequately reviewed and/or approved by senior staff
before release or publication?
Is (was) this review documented?
Were stability data generated and recorded according to GLP
requi rements?
QUALITY ASSURANCE AND ARCHIVING
Have internal QAU inspections been performed for any or all aspects
of the analyses performed for test substance characterization,
assay and stability?
Have these inspections been made part of the QAU inspection record
in the final report?

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Have the raw data for the assay, characterization and stability
related analyses been properly archived according to GLP Stand-
ards regulations?
STANDARD OPERATING PROCEDURES (SOPs)
Are (were) properly approved SOPs in place governing the general
and specific procedures used to generate the test substance
characterization, assay and stability data?
INSTRUMENT LOGBOOKS
Are (were) logbooks in place to record servicing, preventive main-
tenance, calibration and configuration changes for all instruments
in the laboratories used to generate the characterization, stabil-
ity and assay data?
Are all logbooks well organized and clear, and are all data entries
signed and dated?
Are these logbooks being utilized in an effective manner?
PERSONNEL
Are (were) current curricula vitae available for all staff members
involved with the characterization, assay and stability testing?
Are (were) personnel qualified through training or experience to
reliably perform their duties?
GENERAL
If a summary of test substance characterization (may also include assay,
stability and/or other data) has been prepared by the analytical laboratory
or sponsor, it should be thoroughly reviewed as part of the audit and a

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copy obtained for audit report purposes. This internal test substance
report will generally be useful for selection of data to evaluate and audit.
This internal summary, however, may contain proprietary information, and as
such, may be declared confidential.
An official sample may be collected if there is any question regarding
test substance identity, composition, color, properties or state; however,
a photograph or photocopy (of label) will usually be adequate for evidence
purposes.

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IV. FORMULATION OF TEST SUBSTANCE IN CARRIER
Although this study element does not specifically require a chemist
for evaluation, it best lends itself to review by a chemist because of the
nature of the formulating process and the need to assure minimal cross-
contamination during formulating operations.
Formulation of test substance is addressed in 40 CFR Parts 792.47 and
160.47 of the TSCA and FIFRA GLP Standards regulations, respectively. Other
references to test substance formulation can be found in 40 CFR Parts 792.63/
160.63 (Maintenance and Calibration of Equipment), 792.81/160.81 (SOPs),
792.120/160.120 (Protocols) and 792.107/160.107 (Test and Control Substance
Handling).
Appropriate standard operating procedures (SOPs) and the specific study
protocols are important documents tc review before inspecting the formula-
tion- area, interviewing formulating oersonnel or auditing dosage preparation
data.
The following questions should De answered to the chemist's or inspec-
tor's satisfaction, and suspect problems (including SOP and protocol devi-
ations) should be documented accordingly.
ORGANIZATION
How does (did) the formulation section relate organizationally to
the overall laboratory structure?
Who is (was) the supervisor?
What are (were) his or her qualifications in terms of education,
experience and training?

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To whom does (did) the supervisor report?
How large is (was) the formulating staff?
Do (did) they have other responsibilities?
STANDARD OPERATING PROCEDURES (SOPs)
Are the formulation section SOPs complete, up-to-date and properly
approved?
Are they available for everyday staff use?
Are copies of the protocols for all ongoing 6LP regulated studies
maintained in the formulation area.
DIET MIXING DIRECTIONS
How are (were) mixing directions and/or calculations (dosage
levels) transmitted to the formulation section?
Whom do (did) they come from?
Are (were) they transmitted only once at the onset of a study,
regularly throughout the study or only when a formulation change
is (was) necessitated?
Are (were) calculations double-checked in the formulation section
to confirm agreement with target levels?
Are (were) diet-mixing raw data records verified and documented
by the Study Director on a regular basis?

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What is (was) the typical daily routine for diet-mixing, and what
forms are regularly used to prepare dosed diets and other test
substance preparations?
What is the degree of supervision and level of review in the diet-
mixing area?
TEST SUBSTANCE HANDLING
How is (was) the test substance stored prior to use?
Has it been repackaged and/or subdivided from the original
container?
Does labeling meet GLP Standards requirements for all containers?
If stored frozen or refrigerated, is (was) the test substance
container allowed to equilibrate to room temperature in a properly
maintained dessicator prior to opening?
How is (was) the test substance transferred to the weighing con-
tainer or other measuring device?
How is (was) it transferred from the balance to the carrier or
vehicle?
If the test substance is (was) solid and dry-mixed with feed, has
(had) consideration been given to particle size nonuniformity?
DIET PREPARATION
For the study under audit or review, how are (were) the diets
prepared?

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Are (were) they prepared in sequence from low level to high?
Are (were) premixes prepared for any of the dosages?
Are (were) any vehicles or excipients utilized? If so, what?
What was its (their) source(s) and in-laboratory control?
How is (was) the test substance mixed with the carrier?
Is (was) there any grinding, sieving, drying, pelletizing or other
physical treatment of the feed prior to or subsequent to mixing
with test substance?
Are (were) these procedures adequately addressed, described and/or
specified in the appropriate SOP and protocol?
EQUIPMENT
What equipment is (was) used in terms of mixers, grinders, pellet-
izers, balances, scales and so forth?
Are (were) logbooks available and being utilized regularly to
document servicing, preventive maintenance and calibration?
Are (were) SOPs available and being followed for each piece or
each type of equipment?
How are (were) mixers, blenders, sieves, grinders, balances and
pelletizers cleaned between each use to minimize cross-
contamination?
Are (were) any analyses being periodically performed to assure
reliability of the cleaning operations?

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How often have balances and scales been calibrated?
Are these calibrations documented?
Who performed the calibrations?
How often have the balances and scales been professionally
serviced?
Has professional servicing been adequately documented?
DIET STORAGE
Where and how are (were) dosed mixtures and control substances
stored after mixing?
What type or containers and closures are (were) used?
Are (were) containers color-coded or otherwise adequately labeled
to minimize mixup?
Were there expiration dates on the label of each container?
Are (were) chemicals or other materials stored in the immediate
vicinity of the dosed mixtures and controls?
NONFEED MIXTURES
Who is (was) responsible for aqueous-based, gavage and acute test
substance preparations?
Who is (was) responsible for the dose calculations?

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Are (were) the calculations double checked?
How are (were) the dose preparation data recorded?
How soon after preparation are (were) the dosages administered to
the test species?
How are (were) the preparations labeled and stored prior to
administration?
Are (were) there SOPs and protocols available and being followed
regarding these preparations?
WATER-BASED CARRIER
For dosed water preparations, was the stability verified under
actual test conditions (i.e., during exposure to the test species)
so that any possible microbiological degradation was fully
reflected?
Are (were) the water bottles properly cleaned between refillings?
Are (were) the bottles dedicated to a specific dosage level
throughout the study?
Has the water been monitored for consistency of pH, hardness,
residual chlorine and other pertinent parameters?
Have these analyses been properly conducted and documented accord-
ing to sound scientific practice and GLP Standards requirements?
DIET SAMPLING FOR ANALYSIS
At what stage of mixing, how and at what frequency are (were)
samples of dosed and control preparations taken for chemical
analysis?

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Are (were) they taken from the mixer or from the storage container
or during transfer?
Are (were) all samples analyzed?
Are (were) analyses being performed according to the schedule
specified in the protocol?
If pelletization is (was) used, are (were) samples taken for
analyses before or after this step?
Are (were) the analytical samples stored in the formulation area
or analytical laboratory prior to analysis?
How are (were) they stored?
Are (were) storage conditions equivalent to the actual dosed
mixtures?
How are (were) the samples labeled to preclude mixup?
What documentation normally accompanies the samples to the
analytical laboratory?
What are (were) the analytical turn-around times?
How are (were) results transmitted back to the formulation section?
How are (were) problems in mixing resolved?
Are (were) corrective steps taken in a timely enough manner so as
to not compromise the study?
What is (was) the involvement of the Study Director in these
matters?
also Sections IV and V regarding diet analysis for test substance.)

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V. VERIFICATION OF HOMOGENEITY AND STABILITY OF TEST SUBSTANCE IN CARRIER
Homogeneity and stability tests are GLP Standards requirements for
both FIFRA and TSCA with respect to nonclinical health studies, and for TSCA
ecological effects studies only if called for in the specific study protocol.
These two tests are normally performed by or under the direction of the
biological testing facility, but may also be performed by the sponsor or
other contract analytical laboratory. Stability and homogeneity tests are
to be performed prior to or very early in the toxicological study and should
be directed by an approved SOP. Their purpose is to assure that the test
species consistently receive the test substance at the specified dose levels,
[i.e., to verify that poor mixing (stratification, settling, etc.)], decom-
position, or other losses (volatility, precipitation, adsorption, etc.)
have not occurred.
The following questions should be answered satisfactorily either
through examination of raw data and reports, staff interviews and/or labora-
tory visits.
GENERAL
Are (were) there approved and meaningful SOPs covering homogeneity
and stability testing of test substance in carrier?
Were the homogeneity and stability tests performed prior to or
during the early stages of the in-life stage of the toxicological
study?
Were the homogeneity and/or stability tests accomplished on diet
samples prepared in exactly the same manner as the diets prepared
routinely for the in-life portion of the toxicological study?
Were the homogeneity and stability testing performed on the high
and low (not control) dosage levels?

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26
Were at least three different samples (to establish a standard
deviation) taken from the top, middle and bottom of the mixing
hopper or storage bin for the homogeneity test?
Were storage conditions during the stability test identical to or
simi liar to those used during the in-life stages of the toxico-
logical study?
Did the stability test cover at least the maximum period of time
that a prepared diet was used during the study?
METHODOLOGY
Were the analytical methods for homogeneity and stability testing
the same as those used for confirmation of diet levels during the
in-life portion of the feeding study? If not, what were the sig-
nificant differences?
Were the homogeneity and stability analytical methods validated
in terms of precision, accuracy and freedom from interferences?
What reference standards were used in terms of source and quality?
ACCEPTANCE CRITERIA
What criteria have been established and used, if any, to evaluate
the homogeneity and stability analytical data with respect to
acceptability?
What statistics were used, if any?
Were the data acceptable?

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27
ANALYTICAL QUALITY CONTROL
Were the reported analytical results for homogeneity and stability
testing substantiated with supporting quality control data (i.e.,
precision, accuracy and freedom from interference measurements)?
ARCHIVING AND QUALITY ASSURANCE
Were the homogeneity and stability raw data properly archived?
Were the studies and reports inspected and reviewed by the Quality
Assurance Unit?

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VI. CONFIRMATION OF NOMINAL TEST SUBSTANCE LEVELS IN CARRIER
The concentration of the test or control substance shall be determined
"periodically" in the test mixture, as specified in 40 CFR Parts 260.113(a)(1)
and 792.113(a)(1) of the FIFRA and TSCA GLP regulations, respectively.
These analyses are specified for TSCA ecological effects studies [40 CFR
Parts 792.228(f)(1)] only to the extent called for in the specific study
protocol, similar to the requirement for homogeneity and stability testing.
Currently there are no FIFRA ecological effects, chemical fate or chemical/
physical property study GLP regulations in effect.
The term "periodically" is not specifically defined in the TSCA or
FIFRA regulations or in the associated public comment preambles. Generally
these confirmation analyses should be performed at least monthly for long-
term animal studies (>90 days duration) and a minimum of three times at the
beginning, midpoint and conclusion during shorter term studies. Analyses
are to be performed any time the formulation or formulating technique is
modified along with a redetermination of homogeneity and stability, as
appropriate.
The analytical schedule and any changes should be spelled out or des-
cribed in the study protocol and its approved amendments. All dosage levels,
including controls (which should have been prepared in the exact same fashion
as the dosed carrier except for the absence of test substance) should be
analyzed for each periodic determination. Generally, analysis will be pre-
scribed for active principle components only; however, circumstances may
dictate the necessity to determine by-products, metabolites, breakdown
products and/or excipients or vehicles.
During the study audit, the following questions need to be answered
satisfactorily and properly documented if data gaps, discrepancies or poor
practices are evident.

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PROTOCOLS AND SOPs
Is (was) a sampling and analysis schedule spelled out in the study
protocol?
Does (did) the protocol address turn-around time, methodology,
quality control, sampling technique, transfer process (to labora-
tory) and sample storage requirements?
Is (was) there an approved, scientifically sound SOP governing
sampling, sample transfer to the laboratory, sample storage and
reporting of results to the Study Director?
SAMPLING
Have samples been taken that appear to be representative of the
entire batch?
Was each batch sampled if dosage levels were formulated in several
stages?
Were all dosage levels sampled and analyzed, including controls?
Did the analysis represent the final form of the dosed carrier
(i.e., if pelleting was performed, was this the form that was
analyzed)?
If not, was there a rationale for not analyzing the final form?
SAMPLE CONTROL
How are (were) analytical samples labeled and documented so as
to preclude errors during transfer to and within the laboratory?

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Do (did) reported results unequivocally tie in with the labeling
or numbering system, including identification of the specific
study? [Often there may be similar studies with the same chemical
ongoing at the same time (i.e., with rats and mice and/or several
aquatic and avian species), thus, a unique sample identification
is an important factor toward study validity.]
TURN-AROUND TIME AND SAMPLE STORAGE
What are (were) the typical analytical turn-around times during
the beginning, middle and termination of the study? (Document
more extensively, as necessary, particularly if analyses were not
completed and reported before the end of the period for which the
dosed carrier was fed and/or before the end of the period for
which stability was verified).
How are (were) the samples stored and/or preserved, if analyses
were not conducted immediately?
METHODOLOGY
What methodology is (was) being used during the study to analyze
the dosed carrier and controls to confirm target or nominal levels?
Was the method validated for precision, accuracy, specificity,
freedom from interferences and identity of analyte for at least
the high and low dosage levels in the specific carrier utilized
in the study?
Is (was) the method specific for the analyte of interest?
Was the method revalidated for all subsequent diet modifications
or changes made during the study?

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Was the analytical method new for the study or had it been used
for previous studies?
Has the method been formally written up, published or submitted
for publication in a scientific journal?
Is (was) the method the same as employed for stability and homogen-
eity of the test substance in the carrier? (Obtain copy of the
method for reference purposes even though it might not be needed
as an exhibit.)
PERSONNEL
What personnel performed, directed or were otherwise involved
with the analyses?
What were their qualifications in terms of education, training
and experience?
How long had they been in their positions at the time the analyses
were performed?
Was a training plan for technicians, aides and chemists in place?
Was it being followed?
Do the staff members appear to be knowledgeable and interested in
their jobs and duties? Is there a business-like atmosphere to
the laboratory?
ANALYTICAL QUALITY CONTROL
What quality control steps are (were) carried out concurrently
with each batch of analyses to assess precision, accuracy, freedom
from potential interferences, detection limits and confirmation
of analyte identity?

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What acceptance criteria are (were) utilized for evaluating the
reliability of the achieved data?
What was the basis or source of these criteria?
What steps ensued for out-of-specification results in terms of
repeat analyses and/or new dosage preparation?
Was the Study Director notified in a timely manner of all dosage
preparation and/or analytical problems?
ANALYTICAL REPORTS
How are (were) analytical results reported to the Study Director
or other responsible senior study official?
How often?
Was a separate summary prepared for all analytical results that
was not included in the final study report? (Obtain copy.)
Did all internally reported results agree with those given in the
final study report?
If analytical results in the final study report were statistically
summarized (i.e., by listing of means, standard deviations and
significance tests), were these reported values supported by other
internal reports and raw data?
ANALYTICAL REFERENCE STANDARDS
What is (was) the source and date of the analytical reference
standard used for the dosed carrier analyses?

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33
What was the purity of the reference standard?
How and when was the purity established?
Was the standard stored properly (i.e., according to analytical
method or protocol requirements)?
How often are (were) reference standard solutions prepared?
How are reference standard solutions labeled?
Are (were) these preparations documented in the raw data as to
how, when and by whom prepared?
Are (were) the standard preparations described in the method or
an appropriate SOP?
Was linearity of standard response verified over the entire sample
response range?
If not, were sample and standard response typically within a pre-
scribed amount of each other?
Is (was) an internal standard used for the test substance in car-
rier assay?
If so, had it been selected with sound scientific judgment?
Were calculations performed correctly?
Are (were) sample values corrected for recovery or were recoveries
reported separately?

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34
INSTRUMENTATION
Do instruments such as chromatographs, spectrophotometers,
densitometers, balances and so forth appear to be properly main-
tained and operated?
Are logbooks available with each instrument that appear to
accurately portray a historical record of preventive maintenance,
servicing, calibration and changes in configuration?
Do the logbooks reflect proper operation and configuration for
the period of time they were utilized during the audited study?
Does the staff appear knowledgeable regarding the theory, opera-
tion and upkeep of the laboratory equipment?
Are SOPs or operating manuals readily available for routine use
by the instrument operators?
HOUSEKEEPING
What is the overall appearance of the laboratory?
Is it neat and well maintained?
Are reagents, flasks, solutions and other containers well identi-
fied and dated (if necessary)?
ANALYTICAL RAW DATA
Are the analytical values reported in the final study report
fully and accurately supported by the raw data?
Are the raw data well identified as to when, by whom and how they
were generated?

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35
Are the raw data reflective of the methods, techniques and proce-
dures described in the SOPs, protocols and referenced methods?
Are the raw data neat, well organized and easy to follow?
Are chromatograms, spectra, recorder traces, etc. properly iden-
tified, dated and cross-referenced to notebook pages or bench
sheets, as appropriate?
Are all raw data recorded in pen?
Are all corrections properly performed according to GLP Standards
criteria?
Are the raw data archived according to GLP Standards regulations?
QUALITY ASSURANCE
Has the analytical laboratory been regularly inspected by the
quality assurance unit?
Have these inspections been documented in the required QAU inspec-
tion records?
Have these inspections been accurately listed in the study report?
Has the quality assurance unit reviewed and signed the final
report?

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VII. REFERENCES
1.	"Good Laboratory Practice Compliance Inspections of Laboratories
Conducting Health Effects Studies; Inspector's Manual." USEPA, OPTS
Office of Compliance Monitoring, Washington, D.C., April 1985.
2.	Good Laboratory Practices Standards Regulations, 40 CFR Part 160
(FIFRA), 40 CFR Part 793 (TSCA), issued November 29, 1983.
3.	"TSCA Inspection Manual," U.S. EPA, Office of Pesticides and Toxic
Substances, Office of Compliance Monitoring, Washington, D.C.
4.	"Pesticide Assessment Guidelines" (Various health, environmental and
chemical study testing guidelines), U.S. EPA Office of Pesticides and
Toxic Substances, Office of Pesticides, Washington, D.C., 1982 and
updated periodically.
5.	"Environmental Effects Test Guidelines" and "Health Effects Test Guide-
lines," U.S. EPA, Office of Pesticides and Toxic Substances, Office
of Toxic Substances, Washington, D.C., 1982 and updated periodically
[Reprinted in Federal Regulations 50, #188 (September 27, 1985)].
6.	"TSCA Confidential Business Information Security Manual," U.S. EPA,
Office of Toxic Substances, Washington, D.C., November 1985.
7.	"Chemistry for Toxicity Testing," Jameson, C.W. and Walters, D.B.
(Eds.), Butterworth Publishers, Boxton, 1984.
8.	"Principles and Methods of Toxicology (Student Edition)," Hayes, A.W.
(Ed.) Raven Press, N.Y., 1984.
9.	"Managing Conduct and Data Quality of Toxicology Studies," Hoover, B.K.,
Baldwin, J.K. and Uelner, A. (Eds.) Princeton Scientific Publishing
Company, Princeton, NJ, 1986.

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