vvEPA
United States
Environmental Protection
Agency
Office of Pesticides
and Toxic Substances
Washington DC 20460
EPA-560/4-81-002
February 1981
Toxic Substances
Cost Analysis Methodology
and
Protocol Estimates:
OECD Minimum Pre-Market Data
Test Protocols
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EPA-560/4- 81-002
COST ANALYSIS METHODOLOGY
AND
PROTOCOL ESTIMATES:
OECD Minimum Pre-Market Data Test Protocols
by
Janis C. Kurtz
Allan Cohen
Project Officers:
Joye Akridge
Sammy K. Ng
OFFICE OF REGULATORY ANALYSIS
OFFICE OF TOXIC SUBSTANCES
Washington, DC. 20460
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
Washington, DC. 20460
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PREFACE
The attached document is a contractor's study performed
under the supervision and review of the Office of pesticides and
Toxic Substances of the u.S. Environmental Protection Agency.
The purpose of the study is to estimate the cost of performing
certain tests for chemical toxicity and physical/chemical
properties according to protocols developed by the Organization
for Economic Cooperation and Development (OECD).
The OECD tests
considered in this study are those needed to provide the data
included in the OECD Minimum Premarket Data set (MPD).
The MPD is a set of data designed to support premarket
assessment of new chemicals in the twenty-four OECD member
nations.
In January, 1981, EPA published a Federal Register
notice recommending that the MPD be developed by manufacturers of
chemical substances subject to the new chemical notification
program of the Toxic Substances Control Act (TSCA).
This notice
also recommended the use of test protocols from several sources,
including the OECD.
Both the development of the MPD and the use
of the OECD protocols is voluntary.
The cost data in this study,
which represents the first attempt to estimate the cost of
performing
OECD tests in this country, are intended to help
manufacturers make testing decisions.
The OECD test protocols are being made available through the
Industry Assistance Office of the Office of Pesticides and Toxic
Substances (800 424-9065).
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This report was submitted in fulfillment of Task Order
Number 4 of Contract Number 68-01-5864, by Enviro Control, Inc.
Work was completed in January 1981.
The study is not an offica1 EPA publication.
The accompanying
cost estimates represent the views of the contractor who
performed the study.
It cannot be cited, referenced, or
represented in any respect in any such proceedings as a statement
of EPA's view regarding the cost of these protocols.
iv
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TABLE OF CONTENTS
COST ANALYSIS METHODOLOGY
AND
PROTOCOL ESTIMATES:
OECD Minimum Pre-Market Data Test Protocols
INTRODUCTION
CHAPTER I.
I.A.
I.B.
I.C.
I.D.
I.E.
I.F.
I.G.
CHAPTER II.
II .A.
II.B.
II.C.
II.D.
II.E.
II.F.
II.G.
II.H.
11.1.
II.J.
II.K.
II. L.
II.M.
CHAPTER III.
III.A.
III.B.
III.C.
III.D.
III.E.
BASIC COST ESTIMATING PROCEDURES
Introduction
Costing Formula
Direct Labor Cost
Overhead
Other Direct Costs
General and Administrative
Profit or Fee
Cost
PHYSICAL CHEMICAL PROPERTIES
Introduction
Melting Point/Melting Range
Boiling Point/Boiling Range
Density of Liquids and Solids
Vapor Pressure
Water Solubility
Parti tion Coefficient, n - octanol/
water
Hydrolysis (as a function of pH)
Spectra (UV and visible)
Adsorption/Desorption to Soil Types
Dissociation Constant
Particle Size Distribution
Summary
ECOTOXICOLOGY
Introduction
Acute Toxicity LCso, Fish (96 hour,
static)
Reproduction Study and LCso, Daphnia
magna
Growth Inhibition Study, Unicellular
Alga (4 days)
Summary
CHAPTER IV. BIODEGREDATION/ACCUMULATION
IV.A. Introduction
IV.B. Ready Biodegradability
IV.B.l. Closed Bottle Test (Fischer)
IV.B.2. Biodegradability (Microorganisms)
IV.B.3. Biodegradability
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TABLE OF CONTENTS (Continued)
IV.B.4.
IV.B.S.
IV.C.
IV.C.l.
Biodegradability (Sturm test)
Screening Test with DOC Analysis
Bioaccumulation
Bioconcentration in Marine
Organisms
Static Bioaccurnulation in Fish
Summary
IV.C.2.
IV.D.
CHAPTER V. TOXICITY STUDIES FOR HUMAN HEALTH EFFECTS
V.A. Introduction
V.B. Acute Toxicity
V.B.l. Acute Oral Toxicity
V.B.2. Acute Dermal Toxicity
V.B.3. Acute Inhalation Toxicity
V.B.4. Acute Dermal Irritation/Corrosion
V.B.S. Acute Eye Irr~tation!Corrosion
V.B.6. Skin SensitizatiQn
V.c. ~epeated Dose Toxicity
V.D. Mutagenicity
V.D.I. Reyerse Mutption Assay (S. typhimu~ium)
V.D.2. Reve~se Mutatio~ Assay (E. e~Zi WP2)
V.D.3. In V~tpo Mammal~an Cytogenet~cs
V.D.4. In Vivo Mammalian Bone Marrow Cytogenetics
V.D.S. Micronucleus Test
V . E . Summary
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COST ANALYSIS METHODOLOGY
. . A~
PROTOCOL~TIMATES:
OECD Minimum Pre-Market Data Test Protocols
Introduction
The following methodology for estimating the cost of
OECD (Organization for Economic Cooperation and Dev~lopment)
testing protocols has been designed to provide, as completely
as possible, a representative estimate of testing costs in
the United States. The methodology has been developed from
contractor experience, review of previously published
costing methods, and both formal and informal surveys of
other testing laboratories, chemical and pharmaceutical
companies, and prime contractors for Federal testing. programs.
The cost estimating procedures are on a per chemical tested
basis. The accuracy of cost estimates is directly proportional
to the degree of detail specified in each protocol. If certain
protocol details are not specified, then either assumptions
must be made as to the intent of the test author(s), or
broad ranges of component costs must be incorporated into the
cost estimating procedure to encompass the entire range of
protocol possib1ities. These prices are for single tests
only; the total cost of any testing battery will depend upon
which of the tests are performed.
Even if all necessary details of a protocol are clearly
specified, the estimated cost of conducting a study may vary
greatly from laboratory to laboratory.
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Cost estimate variance between testing laboratories
h 2,4,10,31
originates from many sources, suc as:
'. understanding of the nature of the chemical to be tested
. overhead rates
. salary rates
. in-house expertise
. use of outside consultants and subcontractors
. worker productivity and efficiency
. degree of automation
. acquisition of expendable supplies
. marketing strategies
. accuracy of protocol costing procedures
An understanding of the nature of the chemical to be
tested may have a large effect on the time and cost estimated
to conduct the study. Compound preparation and dosing times
are both dependent upon the nature of the test article.
Solid materials may be difficult to maintain in suspension,
requiring a more detailed mixing regimen. Chemical charac-
terisitics may dictate especially frequent sampling to assay
for purity, stability, and concentration; and also dictate
the method by which assays are conducted.
Overhead rates may be significantly different between
testing laboratories due to numerous interacting factors.
The number of years t&at a laboratory has been in operation
may influence the overhead rate. Relatively new laboratories
have a smaller work force (very few of ,which are supported on
overhead), a large capital expenditure for new equipment,
and a sizeable expenditure for marketing to generate new
business. Well established laboratories usually support many
more employees on overhead and spend less on marketing and
new equipment~r
The range and variety of experimental capabilities offered
by a laboratory may also affect the overhead percentage. If
the testing laboratory is one of many operational units, such
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as a testing laboratory associated with a large chemical
manufacturing company, the overhead rate may be inordinately
burdened by cost factors from other operational units, and
is thus difficult to compute accurately.2 The more varied
the laboratory capabilities are, the more equipment and
personnel are required for operation. The more limited
the experimental capabilities, the more the laboratory
must rely on expensive consultants and subcontractors to
perform specialized technical tasks.
The efficiency of the work force is also a variable
factor. Higher salary rates for productive, well-qualified
personnel may balance against lower salaries paid to less
efficient personnel requiring more training and exhibiting
greater turnover. A hi9h degree of automation may boost
worker speed and efficiency, yet the cost of automation
significantly increases the overhead rate; automatic water-
ing systems reduce animal care time, but cause an increased
capital expenditure. A similar situation occurs when
computerized systems are introduced to collect, process,
and store data, which reduces technician and report writer
times.
All expendable supplies required to conduct a study
(e.g., stock, cultures, experimental animals, chemical
reagents, etc.) are subject to many factors which cause
variations in acquisition costs. For example, market
fluctuations and the availability and choice of supplier
directly affect the price of expendable supplies. The
proximity of the testing laboratory to the supplier will
affect shipping costs. Laboratories conducting a high
volume of studies are more likely to take advantage of
volume discounts.
Marketing strategies employed by various laboratories
may also significantly affect the costs of conducting
different protocols. As in most businesses, the overall profit
realized by a testing laboratory is proportional to the volume
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of tests performed. In order to encourage volume testing,
some laboratories will offer discounted prices for multi-
ple testing packages which are significantly lower than
the sum of the costs of the individual components tests.
Low estimates may also be given to solicit new business
when testing volume is low, or when a laboratory wishes to
expand its testing capabilities into new areas.
The accuracy and experience of cost estimating personnel
can also significantly affect estimated costs of specific
protocols. Personnel with little experience may completely
overlook certain cost components or under- or over-estimate
other components. Some laboratories routinely conduct
certain types of studies, while seldom or never conducting
others, even though they have the necessary equipment and
expertise. Cost estimates obtained for the routinely
conducted tests will most likely be accurate, while
estimates for other protocols may show marked variation
from average or actual costs.
Recognizing all of the possible sources of cost
variation, the proposed cost estimating methodology has
been constructed by incorporating best estimates of
industry average figures for all component costs, such as
task times, salaries, overhead rates, supply costs, and
profit margins. Industry averages were estimated by obtaining
this confidential information from testing laboratories,
federal prime contractors, manufacturing companies that
contract similar studies, personnel placement firms,
industry trade associations, and the contractor's own
experience in the field. Despite this effort at designing
an accurate cost estimating methodology, the final estimates
generated are only considered to be representative of the
prices that might actually be charged for conducting a
study in the United States.
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CHAPTER I
BASIC COST ESTIMATING PROCEDURES
LA.
Introduction
Cost estimates are calculated by separating each protocol
into components and estimating the cost of each component. The
major cost components are Direct Labor, Overhead, Other Direct
Costs, General and Administrative Costs and Profit or Fee.
These major areas are subdivided as described below, and
estimated to arrive at a Best Estimate. The cost of most tests
will fall into a range built around the Best Estimate.
In some cases, price quotations and/or cost component re-
quirements were obtc'.ined from several commercial testing
laboratories in lieu of best estimates. Laboratory surveys
were relied upon when the contractor did not have direct
. h d . h 26,28
experience W1t con uct1ng t e test.
Best
Costing Formula
The following components are estimated to provide a
Estimate:
Direct Labor Cost
I.B.
- task identification
- personnel requirements
- task time requirements
- personnel salary rates
Overhead Cost-s
Other Direct Costs
- expendable supplies
General and Administrative
Profit or Fee
Costs
These component costs are combined in the following formula
to calculate the Best ~stimated Cost:
DL + OH + ODC + G&A + F = Best Estimate
where:
DL =
OH =
ODC =
G&A =
Direct Labor Cost
Overhead, as a % of DL
Other Direct Costs
General and Administrative Costs,
as a % of (DL + OH + ODC)
F = Fee, as a % of (DL + OH + ODC + G&A)
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I.C.
Direct Labor Cost
The estimation of direct labor cost involves four
separate considerations:
1)
2)
3)
4)
Task Identification
Personnel Requirements
Task Time Requirements
Personnel Salary Rates
In addition, salary and inflation adjustment factors are
applied if appropriate to account for wage increases likely
to occur during the performance of the study due to inflation
and merit increases.
1.
Task Identification
Each protocol is evaluated by a scientist who
is familiar with the particular type of study, and
the specific tasks required to complete the study are
identified.
2.
Personnel Requirements
The types of professional and technical personnel
required to complete each task are assigned to each
respective task.
The levels of education and experi-
ence required are either specified in the protocol, in
federal regulatory requirements, or are established
by general consensus and practice in the industry.
Examples follow:
Study Director - Ph.D. in toxicology, pharmacology,
or related fields, with a minimum of 4-5 years
experience in toxicology testing. This in-
dividual is responsible for all the overall con-
duct of the study.
Veterinarian - D.V.M.; Board-certified or eligible
for certification by the American College of
Laboratory Animal Medicine with a minimum of
two years of experience in laboratory animal
medicine. This individual oversees all animal
husbandry, veterinary care, and performs com-
plete physical examinations.
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Compound Preparation technician - B.S. degree
in chemistry or other life science and a
minimum of 1-3 years of experience in
analytical or testing laboratories. This
individual is responsible for the prepara-
tion of all test substances for dosing
(e.g., dietary incorporation, dosing solu-
tions, etc.).
Senior Technician - B. S. degree in life sciences,
a minimum of 3 years experience in toxicity
testing, and supervisory skills. This
individual is responsible for the day-to-day
operation of all technical aspects of the
study during the in-life phase. Supervises
Animal Technicians and Caretakers assigned
to the study. Usually certified as an Animal
Technician or Technologist by the American
Association of Laboratory Animal Science
(AALAS) .
Aquatic or Fisheries Biologist - B.S. degree in
Limnology, Ichthyology or other life sciences
and a minimum of 1-3 years experience in
testing laboratories. This individual is
responsible for ascertaining the health status
of all aquatic test animals and supervises
technicians assigned to the study-
Microbiologist, Soil Scientist, Phycologist, Botanist-
B. S. degree in microbiology, geology, phy-
cology, botany or a closely related field as
appropriate to the assigned area of responsi-
bility and a minimum of 1-3 years of exper-
ience in testing laboratories. This
individual performs or directly supervises all
studies as related to the discipline involved.
Analytical Chemist - M. S. or B.S. degree in chem-
istry with 2-4 years of experience in environ-
mental assays. This individual performs
accurate chemical analyses of organic and
inorganic compounds that may be found in bio-
logical systems and is responsible for instr~-
mental techniques required in the analyses
(i.e., gas, liquid chromatography, UV, IR, NMR,
MS, and AA spectrometry).
Analyst - H. S. diploma or its equivalent and a
minimum~f one year of experience in laboratory
testing plus a minimum of three months on-the-
job experience in performing the measurements
being considered.
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Animal Technician - B. S. degree in life science
or equivalent experience in animal testing
laboratories. AALAS-certified or eligible
for Animal Technician. Experienced in
technical skills of dosing, observations,
necropsy, and data collection.
Animal Caretaker - entry-level position. Th.is
individual is responsible for providing
general animal care and husbandry services,
including cage cleaning, feeding, and
watering.
Clinical Laboratory Supervisor - B.S. degree in
life sciences and a minimum of 3 years ex-
perience in clinical laboratory techniques.
Certified as a Medical Technologist or
Clinical Laboratory Technologist by HEW or
the American Society of Clinical Pathology
(ASCP). Oversees all hematology, clinical
chemistry, and urinalysis assays.
Necropsy Supervisor - D.V.M. or equivalent cert-
ification or experience in laboratory animal
surgery and necropsy. This individual over-
sees all necropsies and supervises necropsy
technicians.
Necropsy Technician - A minimum of 1-2 years
experience in animal testing laboratories
and trained in necropsy and tissue trimming
techniques.
Histology Supervisor - ASCP-certified Histotech-
nologist with a minimum of 3 years experience
in histologic processing of tissues. This
individual oversees all histology services
and the production of slides for pathologic
evaluation.
Histology Technician - certified or eligible for
certification by the American Society of
Clinical Pathology (ASCP) with a minimum of
1-2 years experience in histology techniques.
Veterinary Pathologist - Board-certified or
Board-eligible doctoral-level pathologist
with a minimum of 3 years of experience in
veterinary pathology of laboratory animals.
This individual supervises all scheduled
necropsies and performs all microscopic
examinations of tissues.
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Report Writer Supervisor - B.S. in the life
sciences with a minimum of 3 years ex-
perience in toxicological testing and
technical writing. This individual over-
sees the production of all reports of
toxicity studies.
Report Writer - B.S. in life sciences and at
least one year experience in toxicologi-
cal testing or report writing. This
individual compiles, tabulates, and
statistically analyzes test data and writes
materials and methods and results under
the supervision of professionals directing
specific phases of I the study.
Computer Programmer - B. S. in
or equivalent experience.
develops and operates all
used for data collection,
tabulation.
computer science
This individual
computer programs
analyses, and
Computer Coder - a minimum of one year exper-
ience in keypunching.
Report Typist - responsible for all technical
typing involved in the production of final
reports.
General Secretary - provides secretarial support
to scientific and managerial personnel.
Quality Assurance Inspector - B. S. in life
sciences and a minimum of 1-3 years exper-
ience in toxicological testing, quality
control, and/or GLP compliance. This in-
dividual performs all inspections and audits
necessary to insure that a study is per-
formed in compliance with Good Laboratory
Practice.
3.
Task Time Requirements
The time to complete each identified task by the
appropriate personnel is then calculated or estimated.
During the course of a study, the time required
to complete the tasks may change due to familiar-
ity with routines and mortality of test animals.
T.he estimates of task times used in this costing
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methodalogy were obtained from published industry
average figures, surveys of testing laboratories,
and from contractor experience.2,11,31
Personnel Salary Rates
The hourly wage rates paid to each type of pro-
fessional and technical personnel are then multiplied
by the task times to arrive at labor dollars re-
quired to perform each task. The following wage and
salary rates are estimates of current industry
averages obtained from published figures, personnel
placement firms, federal prime contractors, and
. 1 b . 1,11,31
testlng a oratorles:
4.
PERSONNEL
Study Director
Veterinarian
Aquatic Biologist
Microbiologist
Soil Scientist
Phyco1ogist
Botanist
Analytical Che~ist
Analyst
Senior Technician
Animal Technician
Compound Prep. Technician
Animal Caretaker
Clinical Lab. Supervisor
Clinical Lab. Technician
Necropsy Supervisor
Necropsy Technician
Histology Supervisor
Histology Technician
Board-certified Pathologist
Board-eligible Pathologist
Report Writing Supervisor
Report Writer
HOURLY WAGE
$17.80
14.00
6.70
6.70
7.70
6.70
6.70
7.70
4.00
7.70
6.00
6.00
4.00
10.00
6.00
10.00
5.00
10.00
6.00
24.00
19.25
10.00
6.00
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APPROXIMATE
ANNUAL SALARY
$37,000
29,000
14,000
14,000
16,000
14,000
14,000
16,000
8,400
16,000
12,500
12,500
8,400
21,000
12,500
21,000
10,400
21,000
12,500
50,000
40,000
21,000
12,500
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Continued
PERSONNEL
Computer Programmer
Computer Coder
Report Typist
General Secretary
Quality Assurance Inspector
HOURLY WAGE
$ 8.50
5.00
5.00
6.00
10.00
APPROXIMATE
ANNUAL SALARY
$17,700
10,400
10,400
12,500
21,000
Salary Adustment
Direct Labor Dollars are modified by a salary adjustment
factor to account for inflationary and merit wage increases
likely to occur during the performance of studies last-
ing longer than one month. The adjustment factor is
calculated as follows:
Duration of In-life phase (months) x 2
12 months/year x 8% wage increase
per year
The in-life phase is multiplied by 2 because personnel work
on a project until the final report is issued. As a rule
of thumb, the duration of the post mortem phase of the
study is estimated to be as long as the in-life phase. 31
I.D.
Overhead
The overhead rate is applied as a percentage of the total direct
labor cost. This percentage represents a broad spectrum of costs in-
curred in varying degrees by all laboratories performing tests. The
cost of maintaining the laboratory facility, i.e., rental or mortgage,
communication services, utilities, depreciation and amortization of
non-expendable equipment, insurance, property taxes, repairs, and
maintenance are included in overhead. Cost incurred as direct support
are included, i.e., recruitment and relocation of personnel, fees and
licenses, printing and reproduction, publications, office supplies and
materials, postage and delivery, travel and computer expenses.
Fringe benefits are also included in overhead costs. These
benefits may include administrative, sick, and annual leave; holiday
remuneration; group insurance plans (hospitalization, life, disabil-
ity); pension plans; workman's compensation; unemployment and F.I.C.A.
taxes; and tuition reimbursement. The cost of fringe benefits may
vary considerably based on the number of employees receiving benefits
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4
and the range of benefits offered.
Finally, overhead encompasses indirect labor costs, i.e., security
and supervisory labor. Indirect labor provided to perform support
functions, such as standard operating procedures development,
protocol evaluation, and editing of reports, are included in over-
head.
Overhead rates are difficult to obtain from company personnel
due to the confidential nature of financial information. However,
prime contractors for federal testing programs have access to
overhead rates, and private contacts within testing laboratories
sometimes provide estimates. The rates vary considerably, from
50% to 250% of direct labor costs. Taking into consideration the
recent dramatic rise in the cost of energy and the effects of GLP
compliance, an overhead rate of 115% is used in this cost estimating
method as an accurate estimate of the industry average.
I.E.
Other Direct Costs
Other direct costs consist of 1) overtime premium,
2) consultant or subcontracted services and 3) expendable supplies
required to complete the test protocol.
1. Overtime Premium. To comply with test protocols,
designated employees may be required to complete
tasks requiring more time than standard working hours
provide. When the task specified by the test standard
protocol require labor beyond eight hours per day or
40 hours per week, compensating salary may be provided
at one and one-half times the normal hourly rate.
2. Consultant or Subcontracted Services. Since few
laboratories are equipped to perform all tests re-
quired or services dictated by the broad range of
test protocols, laboratories may enlist the aid of
professional consultants or subcontract with other
laboratories' for specific services. These fees are
included in other direct costs. Examples may include
specialized analytical procedures, or veterinary
ophthalmologist services.
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3. Expendable Supplies. Expendable supplies represent
those items ordered and used specifically for the test
protocol. Each protocol is evaluated by an experienced
scientist in order to estimate the dollar amount of
expendable supplies likely to be utilized in conducting
the test standard protocol. This figure does not in-
clude the cost of any major instruments which could be
used in the testing of many different chemicals~l Only
expendables such as experimental animals, reagents,
disposable supplies, and items used specifically for the
test in question are included. For example, to conduct
a partition coefficient assay, a centrifuge is required.
The cost of the centrifuge itself is not included in
other direct costs (although the depreciation and
amortization of this equipment is reflected in overhead
costs), but the reagents necessary to prepare the sample
for centrifugation and any disposable centrifuge tubes
and caps are considered as other direct costs.
Infla-cion, ~~)~~tment - the sum total of all Other Direct
Costs is modified by an inflation factor to account for
increases in the cost of supplies during the course of the
study. This factor is calculated as follows:
Duration of In-Life Phase (months)
x 10% inflation per year
12 months per year
I.F. General and Administrative Cost
These cost represent the salaries of administrative
and support personnel not actually engaged in the study, but
whose labor is necessary to the operation of the laboratory.
Examples of such functions include management, personnel,
purchasing, accounting, marketing, legal counsel, and bid
and proposal staff.3l
As with overhead
rates, G & A rates are difficult to
obtain from private companies.
However, estimates of G & A
~ange fr0m '-'-1- t
25% of the sum of direct labor; overhead,
and other direct costs. An estimated average of 10% is
utilized in this costing methodology.4
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I.G.
Profit or Fee
This figure represents the profit margin applied
to the actual cost of performing a study. Due to the
confidential nature of this type of information, it is
difficult to obtain an accurate survey of profit per-
centages. The range may be from 8% to 35%. An estimated
average of 20% is used in this costing methodology.
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CHAPTER II
PHYSICAL CHEMICAL PROPERTIES
II.A.
Introduction
Through an understanding of the physical and chemical
properties of chemical compounds, a better understanding of
the potential risk presented to man and environment can be
obtained. The method of release to the environment and
therefore the route of human exposure can be better under-
stood by learning the water solubility, density, particle
size distribution, etc. of chemical compounds. An indica-
tion of the mobility inherent in chemical compounds released
in the environment can be ascertained by investigating the
vapor pressure, adsorpt.ion/desorption, etc. of chemical
compounds. Physical and chemical parameters serve also to
indicate further testing necessary for a cO~~lete profile of
the risk associated with chemical compounds.
Variation may result in the reported cost of these
tests if a number of samples are submitted for analysis at
the same time. Discounts are offered by some laboratories
based on the number of samples to be analyzed. A combination
of tests may also cause a reduction in cost. For example,
using the photocell detection method for boiling point/
boiling range determination also allows for measurement
of melting point/melting range. Also, the dynamic method of.
determining boiling point/boiling range also allows for
measurement of vapor pressure, Variation in the cost of
physical chemical properties testing may be as much as 2 fold.
II.B.
Melting Point/Melting Range
The melting point/melting range reflects the tempera-
at which a chemical compound changes in physical state
solid to liquid. Several different methods may be
ture
from
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employed for this determination, including photocell
detection, melting microscope, meniscus method and freezing
point methods. The best estimate for thi~ test is $~OO.
Variance will occur based on the method utilized. The
method to be selected will be determined by the nature of
the chemical compound. Compounds that have high melting
points will require more time and difficulty to test.
Compounds that decompose, show extended softening, react
or volatilize may require more specialized methods and
will entail more cost for analysis.
II.C. Boiling Point/Boiling Range
The boiling pointjboiling range reflects the
temperature at which a chemical compound reaches boiling
temperature. This property indicates the physical state
at which a chemical compound will exist in the environment.
Several different methods may be employed for this determina-
tion including the dynamic method, distillation method and
photocell detection method. The best estimate for this test
is $50. Variance will occur based on the method utilized.
The method to be selected will be determined by the nature
of the chemical compound. Liquids that undergo chemical
reaction below the boiling point, such as oxidation,
rearrangement, or degradation and compounds containing
impurities may require more specialized methods and,
therefore, higher costs.
II.D. Density of Liquids and Solids
The density of a chemical compound is a measurement
of the mass of a unit volume of the compound. This
property may assist in making predictions about the distrib-
tion and settling potential of the compound. Several different
methods may be employed for this determination, which will
be directly dependent on the physical nature of the compound.
The determination of the density of solids
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may be determined by pycnometer, air comparison, pycnometer
or hydrostatic balance. Solids can rise in water or sink
as deposits, becoming incorporated into silt or soil. They
can also enter the atmosphere as particulate dust, which
may later settle as a soil contaminant.
The density of liquids may be determined by hydro-
meter, hydrostatic balance, immersed ball, pycnometer, or
oscillating density meter methods. Determination of
liquids is dependent on viscosity, solubility and inter-
facial tension, which also effect the distribution of
liquids within the environment.
The density of gases may be calculated on the basis
of molecular formula or weight using the gas law. Gases
will settle or become dispersed depending on their density
and also on their diffusion patterns with other molecules
and air turbulence conditions. Since calculation is the
usual method of determining gaseous density, it is not
included in the estimate to follow.
The best estimate for density determinations in solids
and liquids is $50. Variance will occur based on the
method utilized.
II.E. Vapor Pressure
The vapor pressure of a chemical compound is a
measurement of the pressure at which a compound is in
equilibrium between rate of vaporation and condensation.
It is, therefore, an indication of the volatility of a
compound, which provides an understanding of the partition-
ing behavior of a compound. Several different methods
may be employed for this determination including the
dynamic, static, isoteniscopic, vapor pressure balance
and gas saturation methods. The best estimate for this test
is $300. Variation will occur based on the method utilized
and the chemical nature of t~e compound. Compounds with
- 17 -
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low vapor pressure, may sometimes require long sampling times
and, therefore, additional cost.
II.F. Water Solubility
The water solubility of a compound is a measure of
the mass concentration required for saturation in water.
This property indicates the mobility of the chemical
compound within the environment and serves as a prerequi-
site for other tests which will be used for risk assess-
ment. Two different methods may be utilized, depending
on the chemical nature of the compound. Compounds with
low solubilities can be tested with the column elution
method. Compounds with higher solubilities may be deter-
mined with the flask method. The best estimate for this
test is $250. Variance in price will occur based on the
nature of the chemical compound. Determinations for
compounds with low solubilities may be more costly than
those for compounds with high solubilities.
II.G. Partition Coefficient, n-octanol/water
The partition coefficient reflects the equilibrium
concentration ratio of ~n organic chemical compound
as it partitions between a lipophilic solvent {n-octanol}
and water. This property indicates the probable mobility of
chemical compound between water and living organisms. It
also serves as a prerequisite for bioaccumulation deter-
minations since n-octanol simulates fat and will indicate
the storage and ecological magnification potential of
organic chemicals in the fats of living organisms. The
shake flask method is'employed. The two phases are
thoroughly mixed with the compound and allowed to equili-
brate. Both phases are analyzed for compound content.
The hest estimate for this test. is $250. Variance will occur
based on the chemical nature of the compound to be tested.
Surfactants take much more time to equiliQrate. The formation
of emulsions causes problems in reaching equilibrium.
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Metal-organic compounds cannot be assayed with this test,
so other methods must be selected. The chemical analysis
techniques selected to assay the n-octanol and water
partitions will be dictated by the nature of the chemical
compound to be assayed. This factor may cause great
variation in cost.
II.H.
Hydrolysis (as a function of pH)
Hydrolysis is a measurement of the reaction
potential of a chemical with water. The re~ction potential
is determined -as a function of time until a minimum of
90% of the reaction has occurred. This property is an
indication of one degradative pathway a chemical compound
will follow and the potential byproducts that will be
produced in the environment. The hydrolysis is allowed
to take place at four different pH levels and then the
solution is analyzed to determine the per cent reduction
in the initial concentration and the reaction rate
constant is calculated. The best estimate for this test
is $250. Variance will occur based on the analytical
method employed for analysis, which is directly dependent
on the compound to be tested. Unstable compounds may
require specialized methods and could, theref~~e, entail
additional cost.
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Spectra
(UV and Visible)
11.1.
The absorption spectrum of a chemical compound reflects
the particular chemical form of the compound. This property
indicates the potential of a chemical compound to undergo
photochemical degradation and gives an indication of the
byproducts that may be produced within the environment. The
best estimate for this test is $200. This test does not vary
from chemical to chemical substantially, so most costs
encountered should fall within a close range.
II.J.
Adsorption/Desorption to Soil Types
The adsorption/desorption potential of a chemical
compound is a measure of the decrease in concentration that
results when aqueous solutions of the compound are in contact
with different soil types. This property indicates the poten-
tial mobility of the chemical compound within the soil environ-
ment. Three soil types that vary in content and exchange
capacity are utilized. The best estimate for this test is
$2,000. Variability in cost may result based on the sorption
behavior of the compound. Preliminary screening tests of
the nature described may not be comprehensive enough for
adequate determination. Secondary steps may be required
entailing higher costs. Analytical assay of the compound
solution may also be a source of variation in cost. The
analytical method will be dictated by the nature of the
chemical compound.
II.K.
Dissociation Constant
The dissociation constant is a measure of the tendency
of a compound to split into two or more ions. This property
indicates the chemical state or states in which a compound
will exist in the environment. Several different methods may
be employed for this determination including the titration
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method, the spectrophotometric method and conductometric
method. The best estimate for this test is $150. Variance
will occur based on the method utilized. The method to be
selected will be determined by the nature of the chemical
compound. Solubility of the chemical compound will be a
determining factor. The presence of emulsions may require
specialized methods. The analytical methods utilized will
also be determined by the nature of the chemical compound
and may have a great effect on cost.
II. L.
Particle Size Distribution
The particle size distribution of a chemical compound
is a measure of the size and radius distribution of water
insoluble particles and fibers that characterize the compound
This property impacts on the mobility of a chemical compound
in water and air and identifies the potential health hazard
that may arise from inhalation and ingestion of particles and
fibers. Two different methods may be employed. The effec-
tive hydronamic radius determination may be used for particu-
late compounds that do not form fibers. The best estimate
for this test is $100. If fibers do form, microscopic exami-
nation with scanning or transmission electron microscopy is
required for determination. These methods are more costly,
and will increase the best estimated cost. Variability in
cost will also arise based on the chemical nature of the
compound. The occurrence of fiber breakage, clumping or
sample contamination may require more specialized methods
and entail additional cost.
II.M. Summary
The following is a summary of best estimates for
physical/chemical property tests. The estimates listed are
for single tests only. Estimates are not additive, as a
chemical compound may be evaluated using one test, a group
of tests, or none of the tests listed, based on the nature
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of the chemical and its intended use.
Protocol
Melting Point
Boiling Point
Density
Vapor Pressure
Water Solubility
Partition Coefficient
Hydrolysis
Spectra
Absorption/Desorption
Dissociation Constant
Particle Size Distribution
- 22 -
Best Estimate
$ 100
$ 50
$ 50
$ 300
$ 250
$ 250
$ 250
$ 200
$2000
$ 150
$ 100
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CHAPTER III
ECOTOXICOLOGY
III.A.
Introduction
Ecotoxicological investigations provide data necessary
to understand the potential risk a chemical compound poses
as it interacts within ecosystems. Biota representative of
populations and communities within an ecosystem are exposed
to the test compound. This exposure is designed to represent
the actual environmental exposure that will be encountered
by organisms existing in the ecosystem, i.e. in water, soil
or air. The test compound will undergo degradation volatil-
ization, and adsorption as it would within the environment.
Manifestation of toxic effects such as death, reproductive
inhibition, life cycle effects, alteration in growth, and
behavioral changes can be identified. Indications of broader
changes in species diversity or dominance, can be given.
Single species ecotoxicity tests provide necessary pre-
liminary data. Three single species ecotoxicity tests are
presented as requirements for assessing the effects of a
chemical compound on ecosystems. These tests are a Fish
LCso, a Daphnia LCso and an Algal ICso. Data from these
preliminary tests will serve as indicators for further testing
so that a complete profile of the risk produced within eco-
systems can be ascertained.6,8,23,29,30 The variation in
cost for these tests will be minimal, and will arise mainly
from the analytical assay techniques utilized.
III.B.
Acute Toxicity LCso, Fish (96 hour, Static)
This test provides data for determination of the median
lethal concentration (LCso) of a chemical compound in the test
species. This preliminary determination helps to assess the
potential risk of the compound when compared to other known
chemical compounds.
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A range finding test is performed if the toxicity of
the test compound is not already' known. This preliminary (RF)
test establishes the range of test substance concentrations
to be used in the LCso determination. The best estimate
for the range finding test is $150. The best estimate for
the LCso is $400.
Additional costs will' result from these tests based on
the analytical assay techniques employed. Due to the nature
of chemical compounds, different methods will be used and
cos~ will vary. The approximate cost for analytical assay
is $50/sample. Assuming two samples will be required for
the range finding study and 12 samples (two replicates of
six chambers) will be required for the LCso, the total best
estimate for analytical determination is $700.
The chemical nature of the compound may also dictate
other cost variables. If mortality continues beyond 96 hours,
the duration of the test may be extended for adequate
determination of the LC~O' entailing additional cost. Flow
through or static renewal methods may also be utilized,
depending on the nature of the chemical compound. These
methods may entail additional cost. The best estimate for a
Fish LC50 (static) determination is $1250 (RF plus analytical).
III. C.
Reproduction'StudY And LCso, Daphnia magna ,(14 da¥~
This test also allows for determination of LCso, and
also allows for an assessment of reproductive effects since
the test organisms are exposed for more than a complete gen-
eration cycle (three broods).
A range finding test is performed if the toxicity of
the test compound is not already known. This preliminary
test establishes the range of test substance concentrations
to be used in further tests. The best estimate for the range
finding test is $100. The best estimate for the reproduction
study is $600.
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Additional costs will result-from these tests. based on
the analytical assay techniques employed. Due to the nature
of chemical compounds, different methods will be used and
costs will vary. The approximate cost for analytical assay
is $50/samp1e. Assuming two samples will be required for
the range finding study and 12 samples (two replicates of
six chambers) will be required for the reproduction test,
the total best estimate for analytical determination is $700.
Combining the range finding test ($100), the reproduc-
tion test ($600) and the analytical assay ($700) yields a
total best estimate of $1400.
III. D.
Growth Inhibition Study, Unicellular Alga (4 days)
This test provides data for determination of the median
inhibitory concentration (ICso) of a chemical compound with
respect to the test species. This preliminary determination
helps to assess the potential risk presented by a compound.
to primary producers in the environment.
A range finding test is performed if the toxicity of
the test compound is not already known. This preliminary
test establishes the range of test substance concentrations
to be used in the ICso determinations. The best estimate
for the range finding test is $150. The best estimate for
the ICso is $600.
Additional costs will result from these tests if ana-
lytical assay techniques are employed. The test protocol
does not require assay and most laboratories contacted do
not conduct analysis. Due to the nature of chemical com-
pounds, different methods will be used if analysis is conducted
and (costs will vary. The approximate cost for analytical
assay is $50/samp1e. Assuming two samples will be required
for the range finding study and 12 samples (two replicates
of six chambers) will be required for the ICso, the total
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best estimate for analytical determination is $700.
The chemical nature of the compound may also dictate
another cost variables. The water solubility of the com-
pound will have an influence on the method utilized and may
cause some variation in cost.
The best estimate for Algal ICso determination ($600)
including range finding ($150) and analytical assay ($700)
is $1450.
III.E.
Summary
The following is a summary of best estimates for eco-
toxicity testing. The estimates listed are for single tests
only. The total estimates are not additive, as a chemical
compound may be evaluated using one test, a group of tests
or none of the tests listed based on the nature of the chem-
ical and its intended use.
PROTOCOL 23
BEST
ESTH1ATE
TOTAL
ESTIMATE
Toxicity LCso (Fish)
Range Finder
LCso
Analytical Assay
Reproduction Stqdy (Daphnia)
Range Finder
Reproduction test
Analytical Assay
Acute
$1250
$150
400
700
$1400
$100
600
700
Growth Inhibition (Algae)
Range Finder
ICso
Analytical Assay
Without Analytical Assay
$1450
$150
600
700
($ 750)
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CHAPTER IV
BIODEGRADATION/ACCUMULATION
IV.A.
Introduction
Measuring the biodegradation and bioaccumulation
potential of chemicals is important. for assessment of the
hazard presented by chemicals entering the environment.
Five different tests are presented as options for the
assessment of ready biodegradability, and two options
are presented for the assessment of bioaccumulation.
Selection of the appropriate test in each area will de-
pend on the chemical nature of the compound to be tested
and the means by which it will enter the environment.2l
Variation in the cost of these tests will be minimal, and
will arise mainly from adjustments to methods dictated by
the company.
IV.B.
Ready Biodegradability
IV.B.l.
Closed Bottle Test (Fischer Te,st),
This test measures biodegradability based on analysis
of oxygen content. The chemical compound to be tested is
dissolved in an inorganic medium, into which a mixed popu-
lation of microorganisms is inoculated. A period of
incubation follows in closed bottles placed in the dark
in a constant temperature bath. Oxygen analysis over a
28 day period provides an indication of the degradation
occurring. The best estimate for this test is $250. Varia-
bility in cost may result if the compound is difficult to
solubilize into the inorganic medium. Special procedures
to adjust for insolubility will cause an increase in cost.
IV.B.2.
Biodegradability (Microorganisms - MITI Test)
This test measures biodegradability based on bio-
chemical oxygen demand. The chemical compounds to be tested
are solubilized into deionized water, or culture medium with
activated sludge added. Microorganisms are added. Bio-
chemical oxygen demand is measured continuously
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as a means of measuring biodegradability, along with
supplemental chemical analyses. The best estimate for this
test is $9,000. Variability will again result based on the
degree of difficulty presented by solubilizing the test
compound. Volatile compounds require more specialized,
more costly methods.
IV.B.3. Biodegradability (AFNOR Test)
This test measures biodegradability based on dissolved
organic carbon analysis. The chemical com?ounds to be tested
are dissolved in water and inoculated with chemiorganotrophic
microorganism. Organic carbon remaining in solution is
measured at several time intervals concluding with 42 days.
The corresponding level of biodegradation is calculated.
The best estimate for this test is $1,000.00. vari-
ance in cost may be present based on the solubility of the
compound. Methods may require adjustment for volatile
compounds. Concentration of test compounds which inhibit
the growth of microorganisms utilized may also require
adjustments in method, causing additional cost variation.
IV.B.4. Biodegradability (Sturm test)
This test measures biodegradability based on C02
evolution. The chemical compound to be tested is added
to liquid culture medium and inoculated with sewage
microorganisms. The total amount of C02 evolved from the
test system is measured. Biodegradation is calculated
from this result. The best estimate of this test is $1,700.
Variability in cost may be present based on the solubility
of the compound. Insoluble compounds may be utilized as
fine particles in suspension. Modifications to the
compound concentration may be required if the test
compound inhibits the growth of microorganisms utilized.
These method adjustments may cause variation in cost.
IV.B.S. Screening Test With DOC Analysis
This test measures biodegradability based on dis-
solved organic carbon. The chemical compound to be tested
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is dissolved in an inorganic medium and inoculated with
a mixed population of microorganisms. Dissolved organic
carbon is measured over a 28 day period. Biodegradation
is measured as %DOC removed from the test system. The best
estimate for this test is $700. Variability will result
based on the solubility of the test compound. Modifica-
tions to the compound concentration may be required if
the test compound inhibits the growth of microorganisms
utilized. These method adjustments may cause variation
in cost.
IV.C.
Bioaccumulation
IV.C.l.
This
Bioconcentration (Marine Organisms)
test measures uptake of the test compound in
An initial concentration of test substance
mussels.
is administered to mussels in marine aquaria.
The
system is allowed to equilibrate then terminated.
Accumulation within the mussels is determined along with
remaining, steady state concentration within the water.
The best estimate for this test is $850. Variability will result
based on the solubility of the test compound in sea water.
IV.C.2.
Static Bioaccumulation in Fish
This test measures uptake of the test compound in
fish. The test substance is administered to fish in
fresh water aquaria and allowed to equilibrate for 8
days. Water is analyzed for the test compound during
the test and upon termination. At termination the fish
are also analyzed for accumulation of compound. The best
estimate for this test is $2,000. Variance will result based on
the nature of the chemical to be tested. Compound
solubility, volatility, degradability, and polarity will
cause this variability. These factors will determine
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the method of chemical analysis to be used which can
fluctuate greatly in cost.
IV.D. Summary
The estimates for the various options to be considered
available for measuring under the requirement for Ready Biode-
gradability are as follows: Closed Bottle Test (Fischer) -
$250, Biodegradability (Microorganisms) - $9,000, Biodegrada-
bility - $1,000, Biodegradability (Sturm test) - $1,700, and
Screening Test With DOC Analysis - $700, making the range of
cost for Ready Biodegradability $250 - $9,000, depending on
which test is selected.
The best estimate for the options to be considered for
measuring Bioaccumulation are as follows: Bioaccumulation
(Marine Organisms) - $850, Static Bioaccumulation in Fish -
$2,000, making the range of cost for Bioaccumulation $850 -
$2,000, depending on which test is selected.
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CHAPTER V
TOXICITY STUDIES FOR HUMAN HEALTH EFFECTS
V.A.
Introduction
Assessments of physical and chemical properties, eco-
toxicology, and potential for biodegradation and bioaccum-
ulation provide indications of potential hazard presented
by chemical compounds. Toxicity studies as described herein
provide indications of the potential hazard that chemicals
may present to human health. Relatively short term tests
are used to provide a preliminary hazard assessment. The
results may also indicate the need for further studies
necessary to characterize the toxicity and aid in decisions
as to further development of new chemical products.
The tests are designed to examine the relationship
between dose and adverse effects demonstrated, and where
possible, to develop information on the physiological mode
or mechanism of toxic effect.9
The different tests included are designed to take into
account the possible routes of human exposure (oral, dermal,
inhalation) and the possible modes of toxic effect (from
organ system effects to chromosomal aberration). Since
such a variety of effects are possible, flexibility in
experimental design must be present to allow for adequate
detection. Where flexibility exists, variation in cost
is produced. Variation in test procedures will cause variance
in the estimated cost of conducting a study from laboratory
to laboratory. This variation may be as much as 10 fold.
V.B.
Acute ToxicityI2,24,27,30
Acute Oral Toxicityl3
V.B.I.
Acute oral toxicity is measured by administering a
single exposure of the chemical compound by gavage in
graduated doses to ~u1tiple groups o~ experi~ental an!mal$,
with an ob~erv~tion pe~~od of at least 14 days. Toxic
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effects are detected by observation during life and necropsy
after death. The LD50 is statistically determined and used
as a comparison index. Acute oral toxicity is an initial
step in health hazard assessment. The best estimate for
this test is $2,000. Variance will occur based on the
species and strain selected for experimentation, on the
duration of toxic effect, and on the extent of pathology
conducted.
1 . i 14
V.B.2. Acute Derma TO~1C ty
Acute dermal toxicity is measured by administering a
single exposure of the chemical compound directly to the skin
in graduated doses to multiple groups of experimental animals,
wi.th an observatiQn pe~iod of~t .least 14 days. Toxic
effects are detected by observation during life and necropsy
after death. The LD50 is statistically determined and used
as a comparison index. Acute dermal toxicity is used as a
basis to determine potential health hazard from dermal exposure
and may also provide information on dermal absorption potential
and mode of toxic action. The best estimate for this test
is $2,800. This estimate is made with the assumption that
rabbits are the species utilized, and that dosing is to
nonabraded skin using semi-occlusive dressings. Variance
will occur when other species are utilized, when the method
of skin application is different and on the extent of
pathology. conducted.
V.B.3.
. . 15
Acute Inhalation TOX1c1ty
Acute Inhalation Toxicity is measured by administering
a single, defined period exposure (usually 4 hours) of the
chemical compound directly in graduated doses to multiple
groups of experimental animals, with an observation period of
at least 14 days. Toxic effects are detected by observation
during life and necropsy after death. The LC50' median
lethal concentration, is statistically determined and used
as a comparative index. Acute inhalation toxicity is used
as a basis to determine potential health hazard from the in-
halation of volatile, aerosol or suspended particulate sub-
stances. The best estimate for this test is $3,300.
- 32 -
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Variance will occur based on the species and strain selected
for the test, the extent of toxicant delivery checks, the
analytical method used for determination and the extent of
pathology performed.
V.B.4 Acute Dermal Irritation/corrosion16
Acute dermal irritation or corrosion is measured by
administering a single dose of the chemical compound directly
to the skin of experimental animals. Toxic effects are
observed and scored for magnitude. Acute dermal irrita-
tion or corrosion is used as a basis to determine the
possible hazard arising from exposure of the skin to chemical
compounds. The best estimate for this test is $700. Variation
may result based on the species and strain selected and on
the extent of toxicity produced.
V.B.5 Acute Eye Irritation/corrosion17
Acute eye irritation or corrosion is measured by
administering a single dose of the chemical compound directly
to the eyes of experimental animals. Toxic effects are observed
and scored for magnitude. Acute eye irritation or corrosion
is used as a basis to determine the possible hazard
arising from exposure of the skin to chemical compounds. The
best estimate for this test is $450. Variation may result based
on the species and strain selected and on the extent of toxicity
produced.
.. . 18
V.B.6 Skin Sens1t1zat1on
Skin sensitization is measured by exposing experimental
animals to a chemical compound, then administering another
challenge exposure after a period of time. Resultant hyper-
sensitive induction can be detected in this manner. Skin
sensitization is used as a basis to determine the potential
hazard for sensitization produced by repeate~ exposure to
chemical compounds. The best estimate for this test is
variable based on the method employed:
- 33 -
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METHOD
Draize Test
Freund's compiete Adjuvant
Guinea Pig Maximation Test
Split Adjuvant Technique
Buehler Test
CFCAT)
BEST ESTIMATE
$ 6,700
3,900
5,500
3,200
3,500
3,200
3,850
Open Epicutaneous Test
Mauer Optimization Test
V.c.
T . . t. 19, 20
Repeated Dose OX1C1 y
Repeated dose toxicity is measured by administering
repeated doses of a chemical compound by the probable route
of human exposure, in graduated doses to multiple groups of
experimental animals, for a period of 14 to 28 days. Toxic
effects are detected by clinical observation and by clinical
laboratory determinations, necropsy, and histopathologic
assessment. The results provide information to indicate
the potential hazard produced by repeated exposures to
a chemical compound. The best estimate for this test,
based on oral exposure by gavage, is: 14 day exposure,
$10,200; 28 day exposure, $12,800. Variation will occur
based on the species and strain selected for experimentation,
the extent of clinical laboratory determinations, and the
pytp~t of histopathologic assessment performed.
V.D.
Mutagenicity
V.D.l Reverse Mutation Assay (S. typhirnu.riumJ
The reverse mutation assay in SalmoneUa typhil11UY'ium is
a measure of gene mutation produced by chemical compounds in
a histidine requiring strain. Revertant, histidine indep-
endent strains of S. typhirnu.rium are produced by point mutations.
The number of revertant colonies in five strains are compared
to spontaneous revertants in a control system to detect the
presence of increased mutation produced by the chemical
compound. Reverse mutation assay is used as a basis to de-
termine potential human mutagenic agents at the gene level.
The best estimate for this test is $1,000. Variability will
occur based on the number of replicates performed.
- 34 -
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V.D.2
Reverse Mutation Assay (E. aoZi WP2)
The reverse mutation assay in E8aheriahia aoZi WP 2 is
a measure of gene mutation produced by a chemical compound in
a tryptophane requiring strain. Revertant tryptophane inde-
pendent strains of E. aoZi WP 2 are produced by point mutations.
The number of revertant colonies.in three strains are compared
to spontaneous revertants in a control system to detect the
presence of increased mutations produced by the chemical com-
pound. Reverse mutation assay is used as a basis to determine
the potential for mutagenicity and carcinogenicity. The best
estimate for this test is $350. Variability will occur based
on the number of strains utilized and the number of replicates
performed.
V. D. 3 In Vitro Mammalian Cytogenetics
The in vitro cytogenetics test is a measure of chromosomal
a~errations produced in cultured mammalian cells e~posed to
chemical compounds. The exposed cell cultures are treated with
a spindle inhibitor, e.g. clx>lchicine, to arrest cells inmetaphase.
The cells are stained and examined for structural and numerical
aberrations. Structural changes resulting from breakage and
reunion in chromosomes and chromatids as well as numerical changes
resulting in aneuploidy and polyploidy can be detected. The
in vitro cytogenetics test is used as a basis to determine potent-
ial mutagenicity at the chromosome level. The best estimate for
~his test is $3;000. Variability will occur based on the cell
line used for the detection and the method employed.
V.D.4 In Vivo. Mammalian. Bone Marrow Cytogenetics
The in vivo cytogenetics test is a measure of chromosomal
aberration produced in the bone marrow cells of animals exposed
to chemical compounds. The harvested bone marrow cells of ex-
posed animals are treated with colchicine, a spindle inhibitor,
to arrest cells in metaphase. The cells are stained and examined
for structural and numerical aberrations. Structural changes
resulting from breakage and reunion in chromosomes and chromatids
as well as numerical changes resulting in aneuploidy and poly-
- 35 -
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ploidy can be detected. The in vivo cytogenetics test is
used as a basis to determine potential mutagenicity at the
chromosome level. The best estimate for this test is
$13,000. Variability will occur based on the species and
strain utilized for the detection and the method employed.
V.D. 5 Micronucleus Test
The micronucleus test is a measure of chromosomal
aberration produced in the bone marrow cells of animals
exposed to chemical compounds. The harvested bone marrow
cells of exposed animals are stained and scored for the
presence of acentric fragments or chromosomes that lag
behind during the anaphase stage of cell division, or
micronuclei. Polychromatic erythrocytes are the cells
utilized since they lack a nucleus facilitating the
detection of micronuclei. Since micronuclei occur natural-
ly, the quantity of micronuclei associated with increasing.
levels of test compound is compared with positive and neg-
ative controls for assessment. The micronucleus test is
used to determine human mutagenic agents at the chromosome
level. The best estimate for this test is $2,000. Var-
iability will occur based on the species and strain utilized
for the determination and the method utilized.
Summary
V. E.
The following is a summary of best estimates for
toxicity studies performed to investigate human health
effects. The estimates listed are for single tests only.
Estimates are not additive, as a chemical compound may be
evaluated using one test, a group of tests, or none of
the tests listed, based on the nature of the compound and
its intended use.
- 36 -
-------
V.E.
Sununary (con't)
BEST
ESTIMATE
$ 2,000
2,800
3,300
700
450
PROTOCOL
Acute
Acute
Oral Toxicity
Dermal Toxicity
Inhalation Toxicity
Dermal Irritation/Corrosion
Eye Irritation/Corrosion
Sensitization:
Draize Test
Acute
Acute
Acute
Skin
Guinea Pig Maximization
Split Adjuvant Test
Buehler Test
Open Epicutaneous Test
Mauer Optimization Test
Repeated Dose Toxicity (oral gavage)
Test
6,700
3,900
5,500
3,200
3,500
3,200
3,850
FCAT
14 Day Exposure
28 Day Exposure
Reverse Mutation Assay (8. typhirrruriwnJ
Reverse Mutation Assay (E. coli WP 2)
In Vitro Mammalian Cytogenetics
In Vivo Mammalian Bone Marrow Cytogenetics
Micronucleus Test
10,200
12,800
1,000
350
3,000
13,000
2,000
- 37 -
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REFERENCES
lBabco, Eleanor L., Salaries of Scientists, Engineers and
Technicians, 9th Edition, Washington, D.C.:Scientific
Manpower Commission, Nov. 1979.
2Birkner, Frederick E., et.al. "How to Set Up a Computerized
Cost Recovery System For a Large Animal Facility"
LAB ANIMAL: Vol. 9, No.3, May/June, 1980
3 . k
BrUS1C , David and Alfred J.W. Hoorn. Screening Program
for the Identification of Potential Chemical Mutagens
and Carcinogens, Litton Bionetics, August, 1979.
4Financial Analysis of Research and Development Laboratories,
Dun and Bradstreet, 1979.
5Goldstein, Avram, et.al. (eds.). Principles of Drug Action.
N.Y.;N.Y.: John Wiley and Sons, 1974.
6Hedgepeth, Joel W. (ed.). Treatise of Marine Ecology and
Paleoecology, Vol. 1, (The Geological Society of
America, Memoir 67). Boulder, Colorado: Geological
Society of America, 1971.
7
Hodgson, Ernest and Frank E. Guthrie, Introduction to
Biochemical Toxicology. N.Y.;N.Y.: Elsevier North
Holland, 1980.
8Hushon, Judith M., et.al. Information
lation of Toxic Substances, Vols.
Va.: Metrek Division of the Mitre
Required for Regu-
I and II. McLean;
Corporation, June,1978.
9
McRae, Alexander, et.al. (eds). Toxic Substances Control
Sourcebook. Germantown; Md: Aspen Systems Corporation,
1978.
10Miller, Marshall Lee (ed.). Proceedings of the 4th Toxic
Control Conference. Toxic Control, Vol. IV. Washington;
D.C.: Government Institutes, Inc. Feb. 1980.
IlNational Institutes of Health, Division of Research Resources,
Animal Resource Program. Cost Analysis and Rate' Setting
Manual for Animal Resource Facilities. Oct. 1979.
l2NormandY, Mary J. and Robert Reynolds. Acute Toxicity Test-
ing Criteria for Chemical Substances. Washington;D.C.:
U.S. Environmental Protection Agency, July, 1979.
130 . t. f . .
rganlsa lon or EconomlC Cooperatlon and Development. OECD
Guideline for Testing of Chemicals. Appendix 401:
"Acute Oral Toxicity", September 30, 1980.
- 38 -
-------
l40rganisation for Economic Cooperation and Development. OECD
Guideline for Testing of Chemicals. Appendix 402:
"Acute Dermal Toxicity",September 30. 1980
l50rganisation for Economic Cooperation and Development. OECD
Guideline for Testing of Chemicals. Appendix 403:
"Acute Inhalation Toxicity", September 30, 1980.
16 . . f . .
Organ1sat1on or Econom1C Cooperat1on and Development. OECD
Guideline for Testin of Chemicals. Appendix 404:
"Acute Dermal Irr1tation Corros1on', September 30, 1980.
17 . . f .
Organ1sat1on or Econom1C Cooperation and Development. OECD
Guideline for Testin of Chemicals. A endix 405:
"Acute Eye Irritation Corrosion", September 30, 1980
18 . . f .
Organ1sat1on or Econom1C Cooperation and Development. OECD
Guideline for Testing of Chemicals. Aevendix 406:
"Skin Sensitisation", September 30, 1980.
19 . . f . . d 1
Organ1sat1on or Econom1C Cooperat10n an Deve opment. OECD
Guideline for Testing of Chemicals. Appendix 407:
"Repeated Dose Oral Toxicity - Rodent, 28-Day or
J-4-Day Study", September 30, 1980.
200 . . f . . d 1 t
rgan1sat1on or Econom1C Cooperat1on an Deve opmen . OECD
Guideline for Testing of Chemicals. Appendix 408:
"Subchronic Oral Toxicity - Rodent, 90-Day Study",
September 30, 1980.
210rganisation for Economic Cooperation and Development. Expert
Group: Degredation/Accumulation. Final Report, Vols. I and
II., OECDi 1979.
220 . . f . .
rgan1sat1on or Econom1C Cooperat1on and Development. Expert
Group: Physical Chemistry. Final Report. Vo1s. I and II.,
OECDi Dec., 1979
230rganisation for Economic Cooperation and Development. OECD
Report on the Assessmant of Potential Environmental
Effects of Chemicals, Vo1s. I, II, and III, OECD; 1979.
240rganisation for Economic Cooperation and Development. Expert
Group: Short Term and Long Term Toxicology Groups. Final
Report, OECD; Oct.,1979.
250rganisation for Economic Cooperation and Development. Expert
Group: Short Term and Long Term Toxicology Groups. "Principles
for the Evaluation of the Muta enic and Carcino enic
Potent1al 0 C em1ca1s. To yo, 1980.
- 39 -
-------
26press, Jacques Cattel, (ed.). Industrial Research Laboratories
of the United States. 15th Ed. N.Y.:N.Y.: Bowker Co.,
:1.977.
27profile of the Chemical Safety Testing Industry: An Assessment
of Pesticide Testing Capacity. I.C.F., Inc.: May 1980.
28 h lb . h 1 . . .
S e y, M1C ae D., Mutagen1c1ty Test1ng Laboratories.in the
United States. National Institute of Environmental Health
Sciences: April, 1980.
29 . 1 ' Off' f . 'd
U.S. EnV1ronmenta Protect1on Agency. 1ce 0 Pest1c1 e
Programs. Preliminary Report: Cost Analysis of Guidelines
forRegistering Pesticides in the United States. EPA:
January 30, 1978.
30u.s. Environmental Protection Agency. Toxic Substances
Control Act. Premanufacture Testing of Chemical Substances.
Policy Statement. Draft.
31 , 1 '
U.S. Env1ronmenta Protect1on Agency. Support Document. Test
Data Development Standards: Chronic Health Effects.
EPA: May 1979.
- 40 -
-------
50272 -, 0'
REPORT DOCUMENTATION I.L_REPORT NO.
PAGE I EPA-S60 /4- Bl-nn?
I~
3. R8c:ipient's Accession No.
.. 1ft.. and Subtitle
L Report Dete
Cost Analysis Methodology and Protocol Estimates:
OECD Hinirnum Pre-Market Data Test Protocols
..
. January, 1981
,
7. Autl!or(s)
Janis C. Kurtz, Alan Cohen
.. ,..rfO"";nl D,..anization Rept. No.
i. Performinc Orpnization Na.... and AcId-
Enviro-Control, Inc.
Borriston Laboratories, Inc.
11300 Rockville Pike
Rockville, Maryland 20852
10. Pro/8c:tlTasklWottl Unit Ho.
lL ContrwctCC) 0' Grant(G) No.
(C) 68-01-5864'
CG)
II. $paMorinl O,..aniutlon Ha",e end Acid....
U.S. Environmental Protect~9n
OTS/ETD/RIB (TS-779)
401 M Street, S.W.
Washington, D.C. 20460
Agency
13. Type of Report & Period eoo..red
Cost Document for
Recommended Guidelines
I..
IL Supple"'enta", Not..
Project Officers: Joye Akridge and Sammy R. Ng
.11. Allitract CU"'it: 200 -rds)
Tbe purpose of this stQdy is to estimate the cost of the
protocols for the Organization Eor Economic Cooperation and Development
I.!inimum Premarket Data (I.PD). OECD is an international organization
of 24 member nations whose membership includes the major chemical
producers of the non-comm~nist world. The MPD is designed for
premarket assessment of the new chemicals in the OECD member nations.
The base set of premarketing data which EPA is recon~endihg is identioal
to the ~WD ueveloped by the OECD. The protocols were costed by first
breaking them down into the ~arious cost components and then costing
eaCD conponent seperately. The contractor's J:nowledge plus li~ited
outside knowledge from other test labs was used in determining the
cost figures.
(OECD
17. Document ""al)'Wis
.. D8SCrtpto~
Cost Analysis for OECD Minimum Pre-Market Data Test Protocol
.. Identlflers/Open.Ended Terms
DE CD Base Set
Economic Analys$s
Premanufacture Test Guidelines
c. COSAT1 'Fleld/Group
II. Availability Statement
19. Security Class (This Report)
non sensitive
2G. Security Class (ThIs Pale)
Inn",-",,,,,n,,,i +-iup
s.. 'n.tructlon. 011 Reve,.e
%1. No. of Pal"
U. Price
04J.s. GOVERNMENT PRINTING OFFICE: 1981
341-082/232 1-3
OP'T'OHAL fORN 272 (4-
(For",.fly NTlS-35)
o.paft..,.nt of Comme"'"
cs.. ANSl-Z3t.l81
-------
VII""U ..,au,,,
Environmental Protection
Agency
Washington DC 20460
'1'5-799
I V~"gtfv 811\01
Fees paid
Environmental
Protection
Agency
EPA 335
.~
Official Business
Penalty for Private Use $300
First-Class
~
1
EPA 560/4-81-002
------- |