United States
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S8-83-034 Dec. 1983
Project Summary
Quality Control and Quality
Assurance Procedures for
Level 1 Health Effects Bioassays
D. J. Brusick, R. R. Young, B. C. Myhr, and D. R. Jagannath
The purpose of this project was to
develop a quality control (QC) and
quality assurance (QA) document for
the EPA Level 1 environmental assess-
ment biological testing program. This
manual, developed as part of this pro-
gram, supplements the Level 1 biologi-
cal testing procedures manual (1). Al-
though the Level 1 procedures manual
presents detailed information on sample
preparation, bioassay procedures, data
evaluation, and sample ranking, the
Level 1 manual could not readily contain
the detailed procedures for QC and QA.
This manual describes the role and
recommended use of laboratory QC and
QA procedures in the Level 1 biological
testing program.
lERL-RTP's QA audit sample
program is presented in the QC/QA
manual. In this program, audit samples
are made available to laboratories
wishing to verify proficiency in
conducting Level 1 bioassays. Also
described is the documentation
required for test material sampling,
processing, storage, and disposal. The
manual identifies protocol steps for
each Level 1 health effects bioassay and
describes the QC and documentation
procedures required to meet the U.S.
Food and Drug Administration's Good
Laboratory Practice Regulations. In
addition to recommendations for QC
and QA procedures, the document
supplies sample forms which may be
used by laboratories that have not
developed their own standard data
recording forms.
QC and QA procedures vary
somewhat between laboratories, and
this document serves as a model, but
should not be viewed as the only
acceptable approach to QC and QA.
These QC and QA procedures are not
intended to restrict test performance or
improvements in study designs, but are
critical to maintain uniformity of data
generation, documentation, and
interpretation.
This Project Summary was developed
by EPA's Industrial Environmental Re-
search Laboratory. Research Triangle
Park. NC, to announce key findings of
the research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering infor-
mation at back).
Introduction
This document supplements IERL-RTP
Procedures Manual: Level 1 Environmen-
tal Assessment Biological Tests (1). It is a
guide to quality control (QC) and quality
assurance (QA) considerations necessary
to perform Level 1 Health Effects bio-
assays. This report:
1. Supplements the Level 1 manual (1)
with respect to describing standard
operating procedures for QC steps
associated with Level 1 health
effects bioassays.
2. Describes the role and use of
laboratory QA audit samples
prepared to establish uniformity in
laboratories conducting Level 1
health effects bioassays.
3. Describes test material sampling
and processing documentation
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required under Level 1 environmen-
tal assessment biological testing.
4. Describes the protocol steps which
require documentation under the
Good Laboratory Practice (GLP)
Regulations of the U.S. Food and
Drug Administration (FDA).
5. Provides sample forms which can be
used in fulfilling the documentation
requirements.
Recently, the EPA established
requirements for GLP, patterned after the
specifics of the GLP regulations of the
U.S. FDA. GLP regulations require that all
phases of a biological assay be
documented and reviewed by the
assigned individuals. These regulations
also specify that the adequacy of test
materials, facilities, and personnel be
documented. Once the adequacy of the
facility and personnel has been
documented, the GLP regulations
require:
1. A detailed study design describing
the steps involved in the bioassay.
The study design (protocol) must be
supported by laboratory Standard
Operating Procedures (SOPs).
2. A complete set of raw data with
support documentation of medium
and reagent batch (or lot number),
source, and expiration date. All data
must be entered in a permanent
record, signed and dated by the
investigator. Methods for analysis
must also be defined and justified.
3. A report which provides all data
collected in the study. The report
contents must define the study
conditions and must be consistent
with both the original study design
and the raw data.
This manual outlines most of the QC
and documentation steps required to
meet EPA and FDA GLP regulations. QC
and QA procedures are not intended to
restrict test performance or improve-
ments in study designs, but are critical to
maintain uniformity of data generation,
documentation, and interpretation. Each
Level 1 health effects assay is divided into
protocol steps. For each step, the major
operations are identified and the
recommended QC procedures are listed.
Steps which need to be documented are
identified, and sample forms are provided
to assist in the documentation procedure.
Alternate forms can be developed and
used to suit specific laboratory
procedures or protocol modifications.
GLP regulations require that all items
entered on forms be signed (or initialed)
and dated. Any alterations to the
permanent record must be initialed and
dated, and the reason for alteration noted.
Description of Laboratory
Audit Procedures
Laboratories conducting Level 1 health
effects bioassays or intending to initiate
health effects studies are encouraged to
document their proficiency in the health
effects bioassays by participating in the
QA audit sample program. Chapter 2 of
the full report describes the laboratory
performance audit sample program that
was developed as part of lERL-RTP's
environmental assessment testing
program. Audit sample analysis is a direct
and efficient method to help develop,
standardize, and evaluate a laboratory's
proficiency in performing Level 1
bioassays. Coded audit samples will be
supplied to the participating laboratory.
The laboratory will perform Level 1 health
effects bioassays for which it desires to
be audited, using procedures described in
the Level 1 manual (1). The procedure for
requesting QA audit samples is described
in Chapter 2. Procedures are also given
for providing safety data sheets in the
event of an accidental spill or human
exposure to the coded samples.
Test results will be reported to the
EPA/IERL-RTP reference laboratory
which will review the results and prepare
a report evaluating performance of the
qualifying laboratory against:
1. Compliance of study design to pro-
cedures described in the Level 1
manual (1).
2. Development and use of QC/QA
procedures that satisfy GLP
regulations.
3. Agreement of the results and
evaluation of the qualifying labora-
tory with the test data base for each
bioassay developed by the
EPA/IERL-RTP reference laboratory
on the same sample(s).
Description of Sample
Handling and Storage
Procedures
To properly evaluate samples and
reach accurate conclusions regarding
tiieir biological properties, information on
collecting, processing, and shipping the
samples must be transmitted with the I
samples.
The forms in Chapter 3 of the QC/QA
manual provide continuity of the
documentation between the time of
sampling and the time of bioassay. The
forms provide a minimum core of
information necessary for adequate
testing and evaluation of complex envi-
ronmental samples. The first, "Level 1
Sample Collection Form," details howthe
samples are collected and provides basic
information on the sample, sample
source, field storage and sampling
conditions. This information will be
useful in determining the emission rate
for tested samples and will aid in
estimating potential exposure. The
second, "Level 1 Sample Processing
Form," defines the sample type,
processing, and type of bioassay to be
performed on the sample: this form is
completed by the Project Officer for the
environmental assessment. Three other
forms provide a record of test material
receipt, use, and disposition at the bio-
assay test facility.
Description of QC Procedures
and Documentation Required
for the Health Effects Bioassays
Most of the QC/QA manual provides
detailed information on the steps
necessary for adequate QC and
documentation of the Level 1 health
effects bioassays. Chapters 4 through 7
of the full report describe the QC and
documentation considerations as they
apply to each bioassay: the Ames
Sa/Anone/ya/microsome mutagenesis
assay, the Chinese hamster ovary (CHO]
cell clonal toxicity assay, the rabbit
alveolar macrophage (RAM) eel
cytotoxicity assay, and the in vivo roden;
toxicity (WAT) assay.
For convenience, each assay is
organized into discrete procedural steps
A flow diagram of the steps is provided fo
each assay as shown in Figures 1 through
4. In the QC/QA manual, each step ii
developed by summarizing the separati
operations involved and the QC function
associated with the operations and b
defining those steps which need docu
mentation. Sample forms are provided fc
each assay. Usually, documentatio
requirements will be satisfied b
completing data recording forms simile
to those provided for each assay.
Reference
1. Brusick, D. J., R. R. Young. IERL-RT
Procedures Manual' Level 1 Env
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Step
1A
Step
1C
Step 1
Maintain
Cell Cultures
Step 2
Step
2
Treat Test
Cultures
Prepare Test
Cultures
Step 3
Prepare Test
Sample
Step
3
Incubate
Plates
Step 4
Treat Test
Cultures
Step
4
Figure 1. Ames assay steps.
ronmental Assessment Biological
Tests. EPA-600/8-81-024, NTIS
PB 82-228966, Litton Bionetics,
Inc., Kensington, MD, October
1981, 15Opp.
Collect and
Analyze Data
(Plate Counts)
Step 5
Collect
Cytotoxicity Data
Step 6
Evaluate Data
Figure 2. CHO clonal toxicity assay steps.
D. Brusick, R. Young. B. Myhr, and D, Jagannath are with Litton Bionetics, Inc.,
Kensington, MD 2O895.
Raymond G. Merrill is the EPA Project Officer (see below).
The complete report, entitled "Quality Control and Quality Assurance Procedures
for Level 1 Health Effects Bioassays," (Order No. PB84-111 228; Cost: $13.00,
subject to change} will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Industrial Environmental Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
US GOVERNMENT PRINTING OfFICE 1983-659-017/7234
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Step 1
Step 2
Step 3
Step 4
Steps
Step 6
Figure 3.
Step 1
Step 2
Step 3
Collect and Evaluate
Data
Rabbit alveolar macrophage
(RAM) cytotoxicity assay steps.
Step 4
StepS
Care for Animals
Prepare and Administer
Compound
Evaluate Quanta!
Toxicity and
Animal Observations
Evaluate Quantitative
Toxicity and
Animal Observations
Analyze Data and
Designate Toxicity
Figure 4. In vivo rodent toxicity a8$ay~tfe%
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
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