PB84-233295
New and Revised Health
Effects Test Guidelines
October 1984
(U.S.) Environmental Protection Agency
Washington, DC
Oct 84
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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October 1984
PB84-233295
ACUTK EXPOSURE
DERMAL TOXICITY
Ob'KICE Of TOXIC SUBSTANCES
OKKICK OF PESTlCEDEb AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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4. Till, and Subtitle
New and Revised Health Effects Test Guidelines
55 y )
5. Report Date
12.Spon oring Organization N.m. and Address
Lrn( w )OX 9.
13. Type of Report & Period Covered
Annual Report
14.
1 5. Suppiem.ntary Notes
16. Abstract (Umit 200 words)
These documents constitute a set of 2 new health effects test guidelines
as well as 3 existing health effects test guidelines that had been
subjected to major revision. These will be added to (new guidelines)
and serve as substitutes for (revised guidelines) the health effects
test guidelines published by NTIS in October, 1982.
17. Document Analysis a Descriptors
b Identifiers/Open Ended Term,
c COSATI Eieid/Group
18. Availability Statement 19. Secunty Class (This Report) 21. No of Pages
Unclassified 89
Release Unlimited 20. Securut Class (Thus Page) 22. Price
Unclassified
7. Author( a)
9. Psrforrning Organization Name and Address
Office of Pesticides and Toxic Substances
Office of Toxic Substances (TS—792)
United States Environmental Protection Agency
401 tl ST., S.W.
Washington, D.C. 20460
6.
8. Performing Organization Rept No
10. Pro,ect/Task/Wo,k Unit No.
11. Contract(C) or rant(G) Nb
(C)
(G)
(5 .0 ANSI—Z39 18)
Se. Ingt ,jttj ng on Reverse OPTIONAL FORM 272 (4—77)
(Formerly NTIS—35)
Department of Commerce
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TP I3LE OI CONTENTS
HG—l cu te—Derma 1
HG—P cute—Inha1
HG—Acu te—Oral
Most Recent
Date of
Issue
1984
1984
1984
;ibclironic Exposure
formal Toxicity
[ nhalation Toxicity
Oril Toxicity
(‘tironi C I:xposure
(‘h ron ic Toxicity
)ncoqon Cl ty
(‘ombi ned Chronic
‘roXle ’i ty/
)flC()(J(T’fl IC I
I ,I . ‘ tt: I I ’ I ( ‘ ()I (AN /I’ I —IJE
I)crinal Sons it —
zit iOfl
Primary formal
Irri tatAon
I rtni iry i:ye
Err i Lit ion
R(’pro(ItIct— I on/
t ”erti liLy
E 1 feet s
I)ev I opmcntal
roxicit:y Study
lormerly ‘ [ ‘orato(
I riI’ a Iji Lion
I)ovo 1 ()F)mo fl ta •l
‘lox 1 Ci I:y
Pro] Irnhlidry
I)ovel opinontal
ToXicity Screen
Hr—Subchronjc—Derma l
HG—Suhchronic—Inha l
HG—Suhchronjc—Orà l
HG—Chronic
HG—Ch ron ic—Onco
HC,—Chronic—Combjnec-I
‘0X ICETY
IIG—Orcja n/Tissue— De rmal
Serisi t
HG— ( ) rq a n/T4 ss iie— De rm a 1
Irrit
HG—Orqa n/Tissue— Eye
Irrit
IIG—Orga n/Tissue—Repro!
F ’e r t
HG—Organ/Tissue—fey Tox
1983
1983
1983
1983
1983
1983
End e x
(;u idol i nos
• GE N RAE, T )X IC [ [ ‘ TESTING
Acu I Expos ure
l)erjnal Toxicity
Inhalation Toxicity
Oral Toxicity
1982
1983
1982
1983
1984
jenicity HG—Organ/Tissue—Terato, 1q83)
lI(;—organ/Tissuo—nev Tox 1984
Inhal
IIG—Orcjan/Tissue —r)ev Tox
Sc roe n
1984
-------
TABLE OF? CONTENTS
Index
Most Recent
Date of
Issue
(;eno Mutations
Salmonella
yphimurjum
r I a c 5ff
Esch Fa coli WP2
and WP2 ur JA
Aspergil lus
ri idu la ns
Neurospora crassa
Sex Linked
Regess ive
Lethal Test in
Drosoph ii a
inc lanogaster
Somatic Cell in
Culture
MOUSe SpeciEjc
Locus Test
(‘hrornocornal Effects
In Vitro Mammalian
Cytogenet ics
Fri Viva Mammalian
Bone Marrow
Cytotjp net ics
Tests Chromosoma]
Analysis
Fn V Lro Sister
Exchange Assay
In Viva Sister
Chromota Id
Exchange Assay
In Vivo Micro-
nucleus
Heritable Trans—
location Test in
Drosoph ii a
T gaster
Dominant Lethal
Assay
Rodent Heritable
Translocat ion
Assay
HG—Gene Muta—S.
yph imurium
HG-Gene Muta—E.
coli
HG—Gene Muta—A.
nidulans
HG—Gene Muta—N. crassa
HG—Gene Muta—Insects —
HG—Gene Muta—Somatic
Cells
HG—Gene Muta—plammal
HG —Chromo_SiSter_Chromo
In vitro
HG —Chromo_sjster...Chromo
In vivo
1983
1983
1983
1983
HG—Chromo_Mjcronuc 1983
EG—Chromo_Insects 1983
UG—Chromo_Dom Lethal 1983
HG—Chromo_Herit Trans— 1983
locat
Guidelines
[ 1. MIJTA(;F:NI(’Tj.y
1984
1983
1983
HC,—Chromo_in vitro 1983
HG—ChromO.Bone Marrow 1983
1983
1983
II /
-------
TABLE OP CONTENTS
DNA Effects
I)ifterential Growth
Tnhibition of
Repair neficient
Bacteria:
“Bacterial DNA
Damage or Repair
Test s”
Unscheduled DNA
Synthesis
Mammalian Cells
Culture
Mitoic Gene Cortver—
sion in
Saccharomyces
cerevisiae
Most Recent
Date of
Issue
NE(IROTOX [ CITY
Nouropa the logy
Support. Document
Peripheral Nerve
PunCt ion
Support Document
Motc)r Activity
c JI)pOr1 l)ocument
Aciii 1)eIayed
Neurotoxic 1 ty
of Organophos—
ph()ruS Sub—
ta ccc s
Suhchroriic Delayed
NeurotoXiCity of
()rga cop hos pho ru s
Substances
Punctional Obser—
vat lona 1
Battery
Support Document
schedule-Controlled
Operant Behavior
Support Document
HC,—Neuro—Pa th
HS—Neuro—Path
HG—Neuro-Peri Nerve
US—Neuro—Peri Nerve
EG—NeurO--MotOr Nerve
HS—Neuro—Motor Nerve
HG—Neuro—Aôute Delayed
HG—Neuro--SUbChrOfliC
Delayed
HG—Neuro--Battery
HS—Neuro—Battery
HG—Neuro-Operant
Behavior
HS—Neuro—Operaflt
Behavior
1983
1983
1983
l98 3
1983
1983
1983
1983
1983
1983
HG—Spec Stud—Metab
1983
Guide lines
Index
HG—DNA--Damage/Repair
HG—DNA—Unsched Syn
HG—DNA-Gene Conversion
1983
l 83
1983
1983
1983
V. SPEC [ AL STIII)T ES
Metabolism
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— c uce— ye rmai
October 1984
[ . PURPOSE
In the assessment and evaluation of the toxic charac—
teristics of a substance, determination of acute dermal
toxicity is usually an initial step. It provides
information on health hazards likely to arise from short—
term exposure by the derma]. route. Data from an acute
study may serve as a basis for classification and
aheling. It is traditionally a step in establishing a
dosage regimen in subchronic and other studies and may
provide initial information on dermal absorption and the
mode of toxic action of a substance. An evaluation of
dcute toxicity data should include the relationship, if
any, between the animals’ exposure to the test substance
and the incidence and severity of all abnormalities,
including behavioral and clinical abnormalities, the
reversibility of observed abnormalities, gross lesions,
body weight changes, effects on mortality, and any other
toxic effects.
I [ . I)EE INITIONS
A. Acute dermal toxicity is the adverse effects occurring
within a snort time of dermal application of a single
(lose of a substance or multiple doses given within 24
ho urs.
K. Dose is the amount of test substance applied. Dose is
expressed as weight of test substance (g, mg) per unit
weight of test animal (e.g. mg/kg).
C. Dose—response is the relationship between the dose and
the proportion of a population sample showing a defined
e f fec t.
IL [ . APPROACHES TO THE DETERMINATION OF ACUTE TOXICITY
At present, the evaluation of chemicals for acute toxicity
is necessary for the protection of public health and the
environment. When animal testing is required for this
purpose, this testing should be done in ways that minimize
numbers of animals used and that take full account of their
welfare.
EPA recommends the following means to reduce the number of
animals used to evaluate acute effects of chemical exposure
while preserving its ability to make reasonable judgments
about safety:
o Attempt the use of existing data on structurally
related chemicals.
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HG—Ac ute—Dermal
-October 1984
o If data for calculating an LD 50 are needed, perform an
acute toxicity study whereby the value of the data
derived from the investment of animal lives is
enhanced. EPA does not encourage the use of animals
solely for the calculation of an LD 5 O.
o Use methods that minimize the numbers of animals in the
test.
The following provides an expanded discussion of these
Principles and their application to the evaluation of acute
toxicity of chemi als.
Using Data E rom Structurally Related Chemicals . In order
to minimize the need for animal testing, ehe Agency
encourages the review of existing acute toxicity
information on chemical substances that are structurally
related to the agent under investigation. In certain
cases, one may be able to glean enough information from
these surrogate chemicals to make preliminary safety
evaluations that may obviate the need for further animal
testing..
“ Limit” Test . When acute lethality data are desirable,
EPA’s test guideline encourages the use of methods that
minimize the requirement for animals, sometimes by a factor
of 90% as compared to the more traditional LD 50 test. In
the “limit” test, a single group of animals receives a
large dose (2 g/kg body weight) of the agent by the dermal
route. If no lethality is demonstrated, no further testing
for acute dermal toxicity is pursued.
Estimation of Lethal Dose . For those substances
demonstrating lethality in a “limit” test or for substances
for which there are data on structurally related chemicals
that indicate potential acute toxicity below 2 g/kg, the
Agency can use estimates of the dose associated with some
level of acute lethality that are derived from a study
comprising three doses as described in this guideline.
With such an approach, use of greater numbers of animals or
increased numbers of dose levels are not necessary.
Multiple Endpoint Evaluation . The Agency stresses the
simultaneous monitoring of several endpoints of toxicity in
animals in a single acute study including sublethal effects
as well as lethality. Dosed animals are observed for
abnormal behavioral manifestations such as increased
salivation or muscular incoordination, in addition to the
recovery from these effects during the observation
period. Both dead and surviving animals are autopsied to
evaluate gross anatomical evidence of organ toxicity. In
selected cases, additional testing may be justified to
characterize better the kinds of abnormalities that have
been found in the organs of the autopsied animals.
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HG—Ac ute—DermaJ.
October 1984
These sound, scientific practices represent some of the
means which maximize the utility of the data obtained from
a limited number of test animals to achieve a balance
between protecting humans and the environment, and the
welfare and utilization of laboratory animals. When animal
testing is, nonetheless, determined to be necessary to
achieve this balance, the following test method
incorporates the principles discussed above.
lv. PRINCIPLE O THE ‘rEST METHOD
When conducting acute toxicity testing, exposure by dermal
application is recommended for chemicals where exposure of
humans by the dermal route is likely. A single exposure
and a 14—day observation period are used. The test
substance is applied dermally in graduated doses to several
groups of experimental animals, one dose being used per
group. Por the limit test, however, only one group is
tested at a single (high) dose, Subsequent to exposure,
systematic daily observations of effects and deaths are
made. Based on the results of cage—side observations or
gross necropsy, the tester may decide to initiate
histopathological review of certain organs, and/or
additional clinical laboratory tests. Animals that die
during the test are necropsied, and at the conclusion of
Lhe observation period, the surviving animals are
sacrificed and are necropsied.
V. LIMIT TEST
If a test at a dose level of at least 2 g/kg body weight
produces no compound—related mortality, then a study using
Lhcee dose levels will not he necessary.
Vi. TES’r PROCEDURES
A. Animal selection
1. Species and strain
The rat, rabbit or guinea pig may be used. The
albino rabbit is preferred because of its size,
skin permeability and extensive data base.
Commonly used laboratory strains should be
employed. If a species other than the three
indicated above is used, the tester should provide
justification and reasoning for its selection.
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HG_Acute...ljermai
October 1984
2.
Young adult animals Should be used. The following
weight ranges are suggested to provide animals of
a Size which facilitates the Conduct of the
test: rats, 200 to 300 g; rabbits 2.0 to 3.0 kg;
guinea pigs 350 to 450 g.
3. Sex
a. Equal numbers of animals of each sex with
healthy intact skin Should be used for each
dose level.
h. The females should he nulliparous and non—
pregnant
4. NumbersPer seG
At least 5 animals of each sex Should be used at
each dose level.
B. Control groups
A Concurrent untreated control is not necessary. A
vehicle control group should be run concurrently except
when historical data are available to determine the
acute toxicity of the vehicle.
C. Dose levels and dose selection
1. Three dose levels should be used and spaced
appropriately to produce test groups with a range
of toxic effects and mortality rates. The data
should be sufficient to produce a dose— response
curve and permit an acceptable estimation of the
median lethal dose. Range finding studies Using
Single animals may help to estimate the
Positioning of the dose groups so that no more
than three dose levels will he necessary.
2. Vehicle
a. Where necessary, the te t Substance is dissolved
or suspended in a Suitable vehicle. It is
recommended that wherever Possible the use of an
aqueous solution be considered first;” followed by
consideration of a Solution in oil (e.g. corn oil)
and then by Possible solution in other vehicles.
For non—aqueous vehicles the toxic characteristics
of the vehicle Should be known, and if not known
should he determined before the test.
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HG—Ac ute—De rmal
October 1984
h. When testing so].id5, which may be pulverized if
appropriate, the test substance should be
moistened sufficiently with water or, where
necessary, a suitable vehicle to ensure good
contact with skin. When a vehicle is used, the
influence of the vehicle on penetration of skin by
the test substance should be taken into account.
I). Exposure duration
The test substance should be administered over a period
not exceeding 24 hours.
H. Observation period
The observation period should he at least 14 days.
Although 14 days is recommended as a minimum period,
the duration of observation should not be fixed
rigidly. IL should be determined by the toxic
reactions, rate of onset and length of recovery period,
and may thus be extended when considered necessary.
The time at which signs of toxicity appear and
disappear, their duration and the time to death are
important, especially if there is a tendency for deaths
to he delayed.
. Preparation of animal skin
1. Shortly before testing, fur should be clipped from
the dorsal area of the trunk of the test
animals. Shaving may be employed, but it should
he carried out approximately 24 hours before the
test. Care must he taken to avoid abrading the
skin, which could alter its permeability.
2. Not less than 10 percent of the body surface area
should be clear for the application of the test
substance. The weight of the animal should he
taken into account when deciding on the area to be
cleared and on the dimensions of any covering
used.
C. Application of test substance
1. The test substance should be applied uniformly
over an area which is approximately 10 percent of
the total body surface area. With highly toxic
substances the surface area covered may be less,
hut as much of the area should be covered with as
thin and uniform a film as possible.
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HG—Ac ute—Dermal
October 1984
2. The test substance should be held in contact with
the skin with a poro-us gauze dressing and non—
irritating tape throughout a 24—hour exposure
period. The test site should be further covered
in a suitable manner to retain the gauze dressing
and test substance and ensure that the animals
cannot ingest the test substance. Restrainers may
be used to prevent the ingestion of the test
substance, but complete immobil izat ion is not a
recomrne nded method.
3. At the end of the exposure period, residual test
substance should he remaved where practicable
using water or an appropriate solvent.
H. Observation of animals
1. A careful clinical examinationi should be made at
least once each day.
2. Additional observations should be made daily,
especially in the early days of the study.
Appropriate actions should he taken to minimize
loss of animals to the study (e.g. necropsy or
refrigeration of those animals found dead and
isolation of weak or moribund animals).
3. Cage—side observations should include, at the
least, evaluations of the skin and fur, eyes and
mucous membranes, respiratory, circulatory,
autonomic and central nervous systems, somatomotor
activity and behavior pattern. Particular
at tention should be directed to observation of
tremors, convulsions, lethargy, other signs of
central nervous system depression, salivation and
diarrhea.
4. Individual weights of animals should be determined
shortly before the test substance is applied,
weekly thereafter, and at death. Changes- in
weights should be calculated and recorded when
survival exceeds one day.
5. The time of death should be recorded as precisely
as possible.
6. At the end of the test, surviving animals should
be weighed and sacrificed.
I. Gross pathology
A gross necropsy should he performed on all animals
under test. All gross pathology changes should be
recorded.
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u Le— iie L ILIdJ.
October 1984
J. Additional evaluations
In animals surviving 24 hours or more, clinical
chemistry tests or microscopic examination of organs
showing evidence of gross pathology should be
considered because they may yield additional useful
information on the induced toxic effects.
VET. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form, showing for
each test group the number of animals at the start of
the test, body weights, time of death of individual
animals at different dose levels, number of animals
displaying other signs of toxicity, description of
toxic effects and necropsy findings.
B. Evaluation of results
An evaluation of results should include the
relationship, if any, between the dose of the test
substance and the incidence, severity and reversibility
of all abnormalities, including behavioral and clinical
effects, gross lesions, body weight changes, effects on
mortality, and any other toxicological effects.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [ Subpart
J, Part 792, Chapter I of Title 40, Code of Federal
Regulations] the following specific information should
be reported:
1. Tabulation of response data by sex and dose level
(i.e. number of animals exposed; number of animals
showing signs of toxicity; number of animals
dying);
2. Dose—response curves for mortality and other toxic
effects (when permitted by the method of
determination);
3. Description of toxic effects including their time
of onset, duration, reversibility, and
relationship to dose;
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HG—Ac ute—Dermal
October 1984
4. Time of death after dosing;
5. Body weight data;
6. Gross pathology findings; and
7. Histopathology findings and any additional
clinical chemistry evaluations, if performed.
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HG—Acute—Dermal
October 1984
V [ [ T. REF ’ERENCES
[ ‘he following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. This should
not be considered the only source of information on test
performance, however.
1. Bliss, C.I. 1938. The determination of the dosage
mortality curve from small numbers. Quarterly Journal
Phdrm. Pharmacology. 11:192—216.
2. Finney, I).G. 1971. Prohit Analysis. Chapter 3——
Estimation of the median effective dose, Chapter 4——
Maximum ]ikelihood estimation. 3rd Edition. London:
Cambridge University Press. 60 pp.
3. Litchfield, J.T., Jr., Wilcoxon, F. 1949. A
simplified method of evaluating dose—effect
experiemnts, Journal of Pharmacology and Experimental
Therapeutics. 96:99—115.
4. Miller, L.C., Tainter, M.L. 1944. Estimation of the
ED50 and its error by means of logarithmic graph paper,
Proceedings of the Society for Experimental Biology and
Medicine. 57:261—264.
5. NAS. 1977. National Academy of Sciences. Principles
and procedures for evaluating the toxicity of household
substances. Washington, D.C.: A report prepared by
the Committee for the Revision of NAS Publication 1138,
under the auspices of the Committee on Toxicology,
National Research Council, National Academy of
Sciences. 130 pp.
6. Thompson, W.R. 1947. Use of moving averages and
inte rpolat ion to estimate median effective dose.
Bacteriological Review. 11:115—145.
7. Weil, C.S. 1952. Tables for convenient calculation of
median effective dose and instructions in their use.
Biometrics. 8:249—263.
8. WHO. 1978. World Health Organization. Principles and
Methods for Evaluating the Toxicity of Chemicals. Part
I. Environmental Health Criteria 6. Geneva: World
Health Organization. 272 pp.
—9—
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liG—Acute—Inhal
October 1984
ACUTE EXPOSURE
INHALATIoN TOXICITY
OE ”I ”ICE 01’ TOXIC SUBSTANCES
O 1 ’I ”ICE (W PESTICIDES AND TOXIC SUBSTANCES
UNITEI) STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG—Acute—Inhal
October 1984
PURPOSE
in the assessment and evaluation of the toxic character—
istics of a substance that may be inhaled, determination of
acute toxicity is usually an initial step. It provides
information on health hazards likely to arise from short—
term exposure by the inhalation route. Data from an acute
study may serve as a basis for classification and
Iai)elin(J. It is LraditionaUy a step in establishing a
dosage regimen in suhchronic and other studies and may
Provide initial, information on the mode of toxic action of
substance. An evaluation of acute toxicity data should
niclude the relationship, if any, between the animals’
exposure to the test substance and the incidence and
Severity of att abnorma’ities, including behavioral and
clinical abnormatities, the reversibility of observed
dl)normaLities, gross lesions, body weight changes, effects
n mortality, and any other toxic effects.
ii . DE [ NfT [ ONS
A. Acute inhalation toxicity is the adverse effects
caused by a substance following a single uninterrupted
exposure by inhalation over a short period of time (24
hours or less) to a substance capable of being
inhaled.
K. Aerodynamic diameter appl ies to the size of part ides
of aerosols. It is the diameter of a sphere of unit
density which behaves aerodynamically as the particle
of the test substance. It is used to compare
particles of different size and densities and to
predict where in the respiratory tract such particles
may be deposited. This term is used in contrast to
measured or geometric diameter which is representative
of actual diameters which in themselves cannot he
related to deposition within the respiratory tract.
C. The geometric mean diameter or the median diameter is
the calculated aerodynamic diameter which divides the
particLes of an aerosol in half based on the weight of
the particles. 1 ’ifty persent of the particles by
weight will he larger than the median diameter and 50
percent of the particles will be smaller than the
median diameter. The median diameter and its
geometric standard deviation is used to statistically
describe the particle size distribution of any aerosol
based on the weight and size of the particles.
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HG—Ac ute— Inhal
October 1984
D. Inhalable diameter refers to that aerodynamic diameter
of a particle which is considered to be inhalable for
the organism. It is used to refer to particles which
are capable of being inhaled and may he deposited
anywhere within the respiratory tract from the trachea
to the aLveoli. For man, the inhalable diameter is
C’)flS lclere(1 as 15 micrometers or less.
I:. I )SC response is the relationship between the dose (or
concentration) and the proportion of a population
sample showing a defined effect.
Iii. APPROACHES TO THE DETERMINATION OF ACUTE TOXICITY
At present, the evaluation of chemicals for acute toxicity
is necessary for the protection of public health and the
environment. When animal testing is required for this
purpose, this testing should be done in ways that minimize
numbers of animals used and that take full account of their
welfare.
EPA recommends the following means to reduce the number of
animals used to evaluate acute effects of chemicals
exposure while preserving its ability to make reasonable
judgemnents about safety:
Attempt the use of existing data on structurally
related chemicals.
o If data for calculating an LC 50 are needed, perform an
acute toxicity study whereby the value of the data
(lerive(1 I:rom the investment of animal lives is
enhanced. EPA does not encourage the use of animals
solely for the calculation of an LC 50
o Use methods that minimize the numbers of animals in
the test.
[ ‘he following provides an expanded discussion of these
principles and their application to the evaluation of acute
t:oxicity of chemicals.
Using Data From Structurally Related Chemicals . In order
to minimize the need for animal testing, the Agency
encourages the review of existing acute toxicity
information on chemical substances that are structurally
related to the agent under investigation. In certain cases
one may he able to glean enough information from these
surrogate chemicals to make prel iminary safety evaluations
LIiaL may obviate the need for further animal testing.
—2—
-------
HG—Acute—Inhal
October 1984
“ Limit” Test . If a test at an exposure of 5 mg/i (actual
concentration of respirable substances) for 4 hours or,
where this is not possible due to physical or chemical
properties of the test substance, the maximum attainable
concentration, using the procedures described for this
study, produces no compound—related mortality, then a full
study using three dose levels will not be necessary.
F stimation of Lethal Dose . For those substances
demonstrating lethality in a “limit” test or for substances
for which there are data on structurally related chemicals
that indicate potential acute toxicity below 5 mg/i, the
Agency can use estimates of the dose associated with some
level of acute lethality that are derived from a study
compr is ing three doses as described in this guideline.
With such an approach, use of greater numbers of animals or
increased numbers of dose levels are not necessary.
Multiple Endpoint Evaluation . The Agency stresses the
simultaneous monitoring of several endpoints of toxicity in
animals in a single acute study including sublethal effects
as well as lethality. Dosed animals are observed for
abnormal behavioral manifestations such as increased
salivation or muscular incoordination, in addition to the
recovery from these effects during the observation
period. Both dead and surviving animals are autopsied to
evaluate gross anatomical evidence of organ toxicity. In
selected cases, additional testing may be justified to
characterize better the kinds of abnormalities that have
been found in the organs of the autopsied animals.
These sound, scientific practices represent some of the
means which maximize the utility of the data obtained from
a limited number of test animals to achieve a balance
between protecting humans and the environment, and the
welfare and utilization of laboratory animals. When animal
testing is, nonetheless, determined to be necessary to
achieve this balance, the following test method
incorporates the principles discussed above.
—3—
-------
HG—Acute—Inhai
October 1984
1V. PRINCIPLE OF’ THE TEST METHOD
When conducting acute toxicity testing, exposure by
inhalation is recommended for chemicals where exposure of
humans by inhalation is likely. A single exposure and a
14—day observation period are used. The test substance is
administered in graduated doses to several groups of
experimental animals, one dose being used per group. For
the limit Lest, however, only one group is tested at a
single (high) dose. Subsequent to exposure, systematic
daily observations of effects and deaths are made. Based
1fl the results of cage—side observations or gross necropsy,
the tester may decide to initiate histopathological review
of certain organs, and/or additional clinical laboratory
tests. Animal that die during the test are necropsied,
and at the conclusion of the observation period, the
surviving animals are sacrificed and are necropsied.
V. LIMIT TEST
If a test at a dose level of at least 5 mg/i (actual
concentration of respirable substances) for 4 hours or,
where this is not possible due to physical or chemical
properties of the test substance, the maximum attainable
concentration, produces no compound—related mortality, then
a full study using three dose levels will not be necessary.
Vi. TEST PROCEI)URES
A. Animal selection
1. Species and strain
Although several mammalian test species may be
used, the rat is the preferred species. Commonly
used laboratory strains should be employed. ‘If
another mammalian species is employed, the tester
should provide justification and reasoning for
its selection.
2. Age
Young adult animals should he used. The wei ht
variation of animals used in a test should not
exceed ± 20 percent of the mean weight for each
sex.
3. Sex
a. Equal numbers of animals of each sex should
be used for each dose level.
b. The females should be nulliparous and non—
pregnant.
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UG—Acute—Inhal
October 1984
4. Numbers Per Dose Group
At least 5 animals of each sex should be used at
each dose level.
B. Control groups
A concurrent untreated control is not necessary.
Where a vehicle is used to help generate an
appropriate concentration of the substance in the
atmosphere, a vehicle control group should be used
when historical data are not available or adequate to
determine the acute toxicity of the vehicle.
C. Dose levels and dose selection
1. Three exposure concentrations should be used and
spaced appropriately to produce test groups with
a range of toxic effects and mortality rates.
The data should he sufficient to produce a dose—
response curve and permit an acceptable
estimation of the median lethal concentration.
Range finding studies using single animals may
help to estimate the positioning of the test
groups so that no more than three doses will be
necessary.
2. Where necessary, a suitable vehicle may be added
to the test substance to help generate an
appropriate concentration of the test substance
in the atmosphere. If a vehicle or diluent is
needed, ideally it should not elicit important
toxic effects itself or substantially alter the
chemical or toxicological properties of the test
substance.
3. In the case of potentially explosive test
substances, care should be taken to avoid
generating explos ive concentrations.
4. To establish suitable exposure concentrations, a
trial test is recommended.
1). Exposure duration
The duration of exposure should be at least 4 hours
after equilibration of the chamber concentrations.
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UG—Acute—Inha ].
October 1984
E. Observation period
The observation period should be at least 14 days.
Although 14 days is recommended as a minimum period,
the duration of observation should not be fixed
rigidly. It should be determined by the toxic
reactions, rate of onset and length of recovery
period, and may thus be extended when considered
necessary. The time at which signs of toxicity appear
and disappear, their duration and the time to death
are important, especially if there is a tendency for
deaths to be delayed.
V. Inhalation Exposure
1. The animals should be tested with inhalation
equipment designed to sustain a dynamic air flow
o [ 12 to 15 air changes per hour, ensure an
adequate oxygen content of 19 percent and an
evenly distributed exposure atmosphere. Where a
chamber is used, its design should minimize
crowding of the test animals and maximize their
exposure to the test substance. This is best
accomplished by individual caging. As a general
rule to ensure stability of a chamber atmosphere,
the total “volume” of the test animals should not
exceed 5 percent of the volume of the test
chamber. Alternatively, oro—nasal, head—only, or
whole body individual chamber exposure may be
used.
2. A suitable analytical concentration control
system should be used. The rate of air flow
should he adjusted to ensure that conditions
throughout the equipment are essentially the
same. Maintenance of a slight negative pressure
inside the chamber will prevent leakage of the
test substance into the surrounding area.
3. The temperature at which the test is performed
should he maintained at 22°C ( 2°). Ideally,
the relative humidity should be maintained
between 40 to 60 percent, but in certain
instances (e.g. tests on aerosols, use of water
vehicle) this may not be practicable.
G. Physical measurements
Measurements or monitoring should be made of the
following:
1. The rate of air flow should be monitored
continuously, but should be recorded at least
every 30 minutes.
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HG—Acute—Inhaj.
October 1984
2. The actual concentrations of the test substance
should he measured in the breathing zone. During
the exposure period the actual concentration of
the test substance should be held as constant as
practicable. Continuous monitoring is
desirable. Measurement of actual concentrations
should be recorded near the beginning, middle,
and end of the exposure period.
3. During the development of the generating system,
particle size analysis should he performed to
establish the stability of aerosol
concentrations. During exposure, analysis should
he made as often as necessary to determine the
consistency of particle size distribution and
homogeneity of the exposure stream.
4. Temperaturee and humidity should be monitored
continuously but should be recorded at least
every 30 minutes.
H. Food and water during exposure period
Food should he withheld during exposure. Water may
also he withheld in certain cases.
I. Observation of animals
1. A careful clinical examination should be made at
least once each day.
2. Additional observations should he made daily,
especially in the early days of the study.
Appropriate actions should be taken to minimize
loss of animals to the study (e.g., necropsy or
refrigeration of those animals found dead and
isolation of weak or moribund animals).
3. Cage—side observations should include, at the
least, evaluations of the skin and fur, eyes and
mucous membranes, respiratory, circulatory,
autonomic and central nervous systems,
somatomotor activity and behavior pattern.
Particular attention should be directed to
observation of tremors, convulsions, lethargy,
other signs of central nervous system depression,
salivation and diarrhea.
4. Individual weights of animals should be
determined shortly before the test substance is
administered, weekly thereafter, and at death.
Changes in weights should be calculated and
recorded when survival exceeds one day.
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October 1984
5. The time of death should be recorded as precisely
as possible.
6. At the end of the test, surviving animals should
be weighed and sacrificed.
3. Gross pathology
A gross necropsy should he performed on all animals
under test, with particular reference to any changes
in the respiratory tract. Where there are significant
signs of toxicity indicating ‘the possible involvement
of other organs, these should be examined. All gross
pathology changes should be recorded.
K. Additional evaluations
In animals surviving 24 hours or more, clinical
chemistry tests or microscopic examination of organs
showing evidence of gross pathology should be
considered because they may yield additional useful
information on the nature of the induced toxic
effects.
VII . I)ATA ANI) RE PORTING
A. Treatment of results
Data should he summarized in tabular form, showing for
each test group the number of animals at the start of
the test, body weights, time of de th of individual
animals at different dose levels, number of animals
displaying other signs of toxicity, description of
toxic effects and necropsy findings.
B. Evaluation of Results
An evaluation of results should include the
relationship, if any, between the concentration of the
test substance and the incidence, severity and
reversibility of all abnormalities, including
behavioral and clinical effects, gross lesions, body
weight changes, effects on mortality, and any other
toxicological effects.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [ Subpart
J, Part 792, Chapter I of Title 40, Code of Federal
Regulations] the following specific information should
be reported: -
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HG—Ac ute—Inha].
October 1984
1. Test conditions
a. Description of exposure apparatus including
design, type, dimensions, source of air,
system for generating particulates and
aerosols, method of conditioning air,
treatment of exhaust air and the method of
housing the animals in a test chamber.
b. The equipment of measuring temperature,
humidity, and particulate aerosol
concentrations and size be described.
2. Exposure data
These should be tabulated and presented with mean
values and a measure of variability (e.g.
standard deviation) and should include:
a. Airflow rates through the inhalation
equipment;
h. Temperature and humidity of air;
c. Nominal concentration (total amount of test
substance fed into the inhalation equipment
divided by volume of air);
d. Actual concentration in test breathing zone;
and
e. Particle size distribution (e.g. median
aerodymanic diameter of particles with
standard deviation from the mean).
3. Animal data
a. Tabulation of response data by sex and
exposure level (i.e. number of animals
exposed, number of animals showing signs of
toxicity, number of animals dying);
h. Dose—response curves for mortality and other
toxic effects (when permitted by the method
of determination);
c. Description of toxic effects including their
time of onset, duration, reversibility, and
relationship to dose;
13
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HG—Acute—Inhal
October 1984
d. Time of death during or following exposure;
e. Body weight data;
f. Gross pathology findings; and
g. Histopathology findings and any additional
clinical chemistry evaluation, if performed.
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HG—Acute--Inhal
October 1984
V [ tI. REFERENCES
the following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. This should
not be considered the only source of information on test
performance, however.
1. Bliss, C.I. 1938. The determination of the dosage
mortality curve from small numbers. Quarterly Journal
Pharm. Pharmacology. 11:192—216.
2. Finney, D.G. 1971. Probit Analysis. Chapter 3——
Estimation of the median effective dose, Chapter 4——
Maximum likelihood estimation. 3rd Edition.
London: Cambridge University Press. 60 pp.
3. Litchfield, J.T., Jr., Wilcoxon, F. 1949. A
simplified method of evaluating dose—effect
experiments. Journal of Pharmacology and Experimental
Therapeutics. 96:99—115.
4. Miller, L.C., Tainter, M.L. 1944. Estimation of the
ED5O and its error by means of logarithmic graph
paper. Proceedings of the Society for Experimental
Biology and Medicine. 57:261—264.
5. NAS. 1977. National Academy of Sciences. Principles
and procedures for evaluating the toxicity of
household substances. Washington, D.C.: A report
prepared by the Committee for the Revision of NAS
Publication 1138, under the auspices of the Committee
on Toxicology, National Research Council, National
Academy of Sciences. 130 pp.
6. Smyth, H.F., Jr., Carpenter, C.P., Weil, C.S.,
Striegel, J.A. 1962. Range finding toxicity data:
List VI. American Industrial Hygiene Association
Journal. 23:95.
7. Thompson, W.R. 1947. Use of moving averages and
interpolation to estimate median effective dose.
Bacteriological Review. 11:115—145.
8. Weil, C.S. 1952. Tables for convenient calculation
of median effective dose and instructions in their
use. Biometrics. 8:249—263.
9. WHO. 1979. Worici Health Organization. Principles
and Methods for Evaluating the Toxicity of
Chemicals. Part I. Environment Health Criteria 6.
Geneva: World Health Organization. 272 pp.
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f IG—Acute—Ora l
October 1984
ACUTE EXPOSURE
ORAL TOXICITY
OE FICE OF TOXIC SUBSTANCES
OE&ICE OF PESTICIDES AND TOXIC SUBSTANCES
LJNITEJ) STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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KG—Acute—Oral
October 1984
I. PURPOSE
In the assessment and evaluation of the toxic charac-
teristics of a substance, determination of acute oral
toxicity is usually an initial step. It provides
intormation on health hazards likely to arise from short—
term exposure hy the oral route. Data from an acute study
may serve as a basis for classification and labeling. It
is traditionally a step in establishing a dosage regimen in
‘- ubchronic and other studies and may provide initial
n1ormation on the mode of toxic action of a substance. An
evaluation of acute toxicity data should include the
relationship, it any, between the animals’ exposure to the
test substance and the incidence and severity of all
abnormalities, including behavioral and clinical
abnormalities, the reversibility of observed abnormalities,
gross lesions, body weight changes, effects on mortality,
and any other toxic effects.
Ti. I)EEINITIONS
A. Acute oral toxicity is the adverse effects occurring
within a short time of oral administration of a single
dose of a substance or multiple doses given within 24
hours.
H. Dose is the amount of test substance administered.
Dose is expressed as weight of test substance (g, my)
per unit weight of test animal (e.g. mg/kg).
C. Dose—response is the relationship between the dose and
the proportion of a population sample showing a defined
effect.
I [ [ . APPROACHES TO THE DETERMINATION OF ACUTE TOXICITY
At present, the evaluation of chemicals for acute toxicity
is necessary for the protection of public health and the
environment. When animal testing is required for this
purpose, this testing should be done in ways that minimize
numbers of animals used and that take full account of their
we 1 fare.
EPA recommends the following means to reduce the number of
animals used to evaluate acute effects of chemical exposure
while preserving its ability to make reasonable judgments
about safety:
o Attempt the use of existing data on structurally
related chemicals.
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HG—Acute—Oral
October 1984
o If data for calculating an LDSO are needed, perform an
acute toxicity study whereby the value of the data
derived from the investment of animal lives is
enhanced. EPA does not encourage the use of animals
solely for the calculation of an LD 50 .
o Use methods that minimize the numbers of animals in the
test.
The following provides an expanded discussion of these
principles and their application to the evaluation of acute
toxicity of chemicals.
Using 1)ata rorn Structurally Related Chemicals . In order
to minimize the need for animal testing, the Agency
encourages the review of existing acute toxicity
information on chemical substances that are structurally
related to the agent under investigation. In certain cases
one may be able to glean enough information from these
surrogate chemicals to make preliminary safety evaluations
that may obviate the need for further animal testing.
“ Limit” Test . When acute lethality data are desirable,
EPA’s test guideline encourages the use of methods that
minimize the requirement for animals, sbmetimes by a factor
of 90% as compared to the more traditional LD 50 test. In
the “limit” test, a single group of animals is given a
large dose (5 g/kg body weight) of the agent. If no
etha1 ity is demonstrated, no further testing for acute
oral toxicity is pursued.
Estimation of Lethal Dose . Fat those substances
demonstrating lethality in a “limit” test or for substances
tor which there are data on structurally related chemicals
that indicate potential acute toxicity below 5 g/kg the
Agency can use estimates of the dose associated with some
level of acute lethality that are derived from a study
comprised of three doses as described in this guideline.
With such an approach, use of greater numbers of animals or
increased numbers of dose levels are not necessary.
Multiple Endpoint Evaluation . The Agency stresses the
simultaneous monitoring of several endpoints of toxicity in
animals in a single acute study including sublethal effects
as well as lethality. Dosed animals are observed for
abnormal behavioral manifestations such as increased
salivation or muscular incoordination, in addition to the
recovery from these effects during the observation
period. Both dead and surviving animals are autopsied to
evaluate gross anatomical evidence of organ toxicity. In
selected cases, additional testing may be justified to
characterize better the kinds of abnormalities that have
been found in the organs of the autopsied animals.
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October 1984
These sound, scientific practices represent some of the
means which maximize the utility of the data obtained from
a limited number of test animals to achieve a balance
between protecting humans and the environment, and the
welfare and utilization of laboratory animals. When animal
testing is, nonetheless, determined to be necessary to
achieve this balance, the following test method
1.ncorl)orates the principles discussed above.
IV. PRINCIPLE OI ’ THE rEsT METHOD
When conducting acute toxicity testing, exposure by gavage
is recommended for chemicals where exposure of humans by
t-he oral route is likely. A single exposure and a 14—day
observation period are used. The test substance is
administered orally in graduated doses to several groups of
experimental animals, one dose being used per group. For
the limit test, however, only one group is tested at a
single (high) dose. Subsequent to exposure, systematic
(laily observations of effects and deaths are made. Based
on the results of cage—side observations or gross necropsy,
the tester may decide to initiate histopathological review
of certain organs, and,/or additional clinical laboratory
tests. Anirials that die during the test are necropsied,
and at the conclusion of the observation period, the
surviving animals are sacrificed and are necropsied.
V. LIMIT TEST
If a test at a dose level of at least 5 g/kg body weight
produces no compound—related mortality, then a study using
three dose levels will not be necessary.
VI. TEST PROCEDURES
A. Animaj. selection
1. Species and strain
Although several mammalian test species may be
used, the rat is the preferred species. Commonly
used laboratory strains should he employed. If
another species is used, the tester should provide
justification and reasoning for its selection.
2. Age
Young adult animals should he used. The weight
variation of animals used in a test should not
exceed ± 20 percent of the mean weight for each
sex.
3 25
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HG—Ac ute—Oral
October 1984
3. Sex
a. Equal numbers of animals of each sex should
he used for each dose level.
h. The females should be nulliparous and non—
pregnant.
4. Numbers Per Dose Group
At least 5 animals of each sex should be used at
each dose level.
K. Control groups
A concurrent untreated control is not necessary. A
vehicle control group should be run concurrently except
when historical data are available to determine the
acute toxicity of the vehicle.
C. Dose levels and dose selection
1. Three dose levels should be used and spaced
appropriately to produce test groups with a range
of toxic effects and mortality rates. The data
should be sufficient to produce a dose— response
curve and permit an acceptable estimation of the
median lethal dose. Range finding studies using
single animals may help to estimate the
positioning of the dose groups so that no more
than three dose levels will be necessary.
2. Vehicle
Where necessary, the test substance is dissolved
or suspended in a suitable vehicle. It is
recommended that wherever possible the use of an
aqueous solution be considered first, followed by
consideration of a solution in oil (e.g. corn oil)
and then by possible solution in other vehicles.
For non—aqueous vehicles the toxic characteristics
of the vehicle should be known, and if not known
should he determined before the test.
3. Volume
The maximum volume of liquid that can be
administered at one time depends on the size of
the test animal. In rodents, the volume should
not exceed 1 mi/lOU g body weight, except when an
aqueous solution is used where 2 ml/lOO g may be
administered. Variability in test volume should
be minimized by adjusting the concentration to
ensure a constant volume at all dose levels.
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October 1984
D. Exposure duration
The test substance should he administered over a period
not exceeding 24 hours.
E. Observation period
The observation period should he at least 14 days.
Although ]4 days is recommended as a minimum period,
the duration of observation should not be fixed
rigidly. It should be determined by the toxic
reactions, rate of onset and length of recovery period,
and may thus be extended when considered necessary.
The time at which signs of toxicity appear and
disappear, their duration and the time to death are
important, especially if there is a tendency for deaths
to be delayed.
F. Exposure
1. The test substance should be administered in a
single dose by gavage, using a stomach tube or
suitable intubation cannula.
2. Animals should be fasted prior to test substance
administration. For the rat, food should be
withheld overnight; for other rodents with higher
metabolic rates a shorter period of fasting is
appropriate.
3. After the substance has been administered, food
may be withheld for an additional 3—4 hours.
4. If a single dose is not possible, the dose may be
given in smaller fractions over a period not
exceeding 24 hours. Where a dose is administered
in fractions, it may be necessary to provide the
animals with food and water depending on the
length of the dosing period.
G. Observation of animals
1. A careful clinical examination should be made at
least once each day.
2. Additional observations should be made daily,
especially in the early days of the study.
Appropriate actions should be taken to minimize
loss of animals to the study (e.g. necropsy or
refrigeration of those animals found dead and
isolation of weak or moribund animals).
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October 1984
3. Cage—side observatiogns should include, at the
least, evaluation of the skin and fur, eyes and
mucous membranes, respiratory, circulatory,
auton rnic and central nervous systems, somatomotor
act iv ity and behavior pattern. Particular
attention should he directed to observation of
tremors, convulsions, lethargy, other signs of
central nervous system depression, salivation and
diarrhea.
4. Individual weights of animals should be determined
shortly before the test substance is administered,
weekly thereafter, and at death. Changes in
weights should be calculated and recorded when
survival exceeds one day.
5. The time of death should be recorded as precisely
as possible.
6. At the end of the test, surviving animals should
he weighed and sacrificed.
H. Gross pathology
A gross necropsy should he performed on all animals
under test. All gross pathology changes should be
recorded.
I. Additional evaluations
In animals surviving 24 hours or more, clinical
chemistry tests or microscopic examination of organs
showing evidence of gross pathology should be
considered because they may yield additional useful
information on the nature of the induced toxic effects.
V [ I. DATA AND REPORTING
A. Treatment of results
Data should he summarized in tabular form, showing for
each test group the number of animals at the start of
the test, body weights, time of death of individual
animals at different dose levels, number of animals
displaying other signs of toxicity, description of
toxic effects and necropsy findings.
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HG—Ac ute—Oral
October 1984
3. Evaluation of results
An evaluation of results should include the
relationship, if any, between the dose of the test
substance and the incidence, severity and reversibility
of all abnormalities, including behavioral and clinical
effects, gross lesions, body weight changes, effects on
mortality, and any other toxicological effects.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [ Subpart
3, Part 792, Chapter I of Title 40, Code of Federal
Regulations} the following specific information should
be reported:
1. Tabulation of response data by sex and dose level
(i.e. number of animals exposed; number of animals
showing signs of toxicity; number of animals
dying);
2. Dose—response curves for mortality and other toxic
effects (when permitted by the method of
determination);
3. Description of toxic effects, including their time
of onset, duration, reversibility, and
relationship to dose;
4. Time of death after dosing;
5. I3ody weight data;
6. Cross pathology findings; and
7. Histopathology findings and any additional
clinical chemistry evaluations, if performed.
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HG—Ac ute—Oral
October 1984
VIII. REFERENCES
[ ‘he following references may be helpful in developing
acceptable protocols, and provide a background of
intormation on which this section is based. This should
not be considered the only source of information on test
performance, however.
1. Balazs, T. 1970. “Measurement of acute toxicity,” in
“Methods in Toxicology.” Edited by G.E. Paget.
Philadelphia: F.A. Davis Co. PP. 49—82.
2. Bliss, C.I. 1938. The deteirmination of the dosage
mortality curve from small numbers. Quarterly Journal
Pharm. Pharmacology. 11:192—216.
3. Finney, D.G. 1971. Probit Analysis. Chapter 3——
Estimation of the median effective dose, Chapter 4——
Maximum likelihood estimation. 3rd Edition. London:
Cambridge University Press. 60 pp.
4. Hunter, W.J .,, Lingk,W.,, Recht, P. 1979.
Intercomparison study on the determination of single
administration toxicity in rats. Journal Association
of Official Analytical Chemists. 62(4):864—873.
5. Litchfield, J.T., Jr., Wilcoxon, F. 1949. A
simplified method of evaluating dose—effect
experiments, Journal of Pharmacology and Exper imental
Therapeutics. 96:99—115.
6. Miller, L.C., Tainter, •M.L. 1944. Estimation of the
ED5O and its error by means of logarithmic graph paper,
Proceedings of the Society for Experimental Biology and
Medicine. 57:261—264.
7. NAS. 1977. National Academy of Sciences. Principles
and procedures for evaluating the toxicity of household
substances. Washington, D.C.: A report prepared by
the Committee for the Revision of NAS Publication 1138,
under the auspices of the Committee on Toxicology,
National Research Council, National Academy of
Sciences. 130 pp.
8. Thompson, W.R. 1947. Use of moving averages and
interpolation to estimate median effective dose.
Bacteriological Review. 11:115—145.
30
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October 1984
9. Weil, C.s. 1952. Tables for convenient calculation of
median effective dose and instructions in their use.
Biometrics. 8:249—263.
10. WHO. 1978. World Health Organizatjo Principles and
Methods for Evaluating the Toxicity of Chemicals. Part
I. Environmental Health Criteria 6. Geneva: World
Health Organizatj 272 pp.
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HG-Organ/Tissue_DeW Tox
October 1984
DEVELOPMENTAL TOXICITY STUDY
Office of Toxic Substances
Office of Pesticides and Toxic Substances
United States Environmental Protection Agency
Washington, D.C. 20460
,32
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HG—Organ/Tissue--Dev Tox
October 1984
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of a chemical, determination of the
potential developmental toxicity is important. The
developmental toxicity study is designed to provide
information on the potential hazard to the unborn
which may arise from exposure of the mother during
pregnancy.
II. DEE ’rNFrIoNs
A. Developmental toxicity is the induction of adverse
effects on the developing organism as a result of
in utero exposure to an agent. It is a generic
term which includes endpoints such as resorptions,
structural abnormalities, growth retardation as
well as functional and behavioral deficits.
B. Dose is the amount of test substance administered.
Dose is expressed as weight of test substance
(g, mg) per unit weight of a test animal (e.g.
mg/kg).
C. No—observed—effect level is the maximum
concentration in a test which produces no
observed adverse effects. A no—observed—effect
level is expressed in terms of weight of test
substance given daily per unit weight of test
animal (mg/kg).
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October 1984
[ II. PRINCIPLE OF THE TEST METHOD
The test substance is administered in graduated
doses for at least that part of the pregnancy
covering the major period of organogenesis, to several
groups of pregnant experimental animals, one dose level
being used per group. Shortly before the expected date
of delivery, the pregnant females are sacrificed, the
uteri removed, and the contents examined for embryonic or
fetal deaths, and live fetuses.
IV. LIMIT TEST
If a test at an exposure of at least 1000 mg/kg body
weight, using the procedures described for this study,
produces no observable developmental toxicity, then a full
study using three dose levels might not be necessary.
V. TEST PROCEDURES
A. Animal selection
1. Species and strain
Testing should be performed in at least 2
mammalian species. Commonly used species
include the rat, mouse, rabbit, and
hamster. If other mammalian
species are used, the tester should provide
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RG—Orgart/Tissue—Dev Tox
October 1984
justification/reasoning for their
selection. Commonly used laboratory strains
should be employed. The strain should not
have low fecundity and should preferably be
characterized for its sensitivity to
developmental toxins.
2. Age
Young adult animals (nulliparous females)
should be used.
3. Sex
Pregnant female animals should be used at
each dose level.
4. Number of animals
At least 20 pregnant rats, mice or hamsters
or 12 pregnant rabbits are recommended at
each dose level. The objective is to
ensure that suEficient pups are produced to
permit meaningful evaluation of the potential
developmental toxicity of the test substance.
B. Control group
A concurrent control group should be used. This
group should he an untreated or sham treated
control group, or, if a vehicle is used in
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HG—Organ/TisSUe—DeV Tox
October 1984
administering the test substance, a vehicle control
group. Except for treatment with the test
substance, a nimals in the control group(s) should
he handled in an identical manner to test group
animals.
C. Dose levels and dose selection
1. At least 3 dose levels with a control and,
where appropriate, a vehicle control, should
be used.
2. The vehicle should neither be developmentally
toxic nor have effects on reproduction.
3. To select the appropriate dose levels, a
pilot or trial study may be advisable.
It is not always necessary to carry out a
trial study in pregnant animals. Comparison
of the results from a trial study in non—
pregnant, and the main study in pregnant
animals will demonstrate if the test
substance is more toxic in pregnant animals.
If a trial study is carried out in pregnant
animals, the dose producing embryonic or
fetal lethalities or maternal toxicity should
be determined.
4. Unless limited by the physical/chemical
nature or biological properties of the
substance, the highest dose level should
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October 1984
induce some overt maternal toxicity such
as weight loss or decrease in weight gain,
but not more than 10 percent maternal deaths.
5. The lowest dose level should not produce any
grossly observable evidence of either
maternal or developmental toxicity.
6. Ideally, the intermediate dose level(s)
should produce minimal observable toxic
effects. If more than one intermediate
concentration is used, the concentration
levels should be spaced to produce a
gradation of toxic effects.
1). Observation period
Day 0 in the test is the day on which a vaginal
plug and/or sperm are observed. The dose period
should cover the period of major organogenesis.
This may be taken as days 6—15 for rat and mouse,
6—14 for hamster, or 6—18 for rabbit.
E. Administration of test substance
The test substance or vehicle is usually
administered orally, by oral intubation unless
the chemical or physical characteristics of the
test substance or pattern of human exposure suggest
a more appropriate route or administration.
The test substance should be administered at the
same time each day.
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October 1984
Exposure conditions
The female test animals are treated with the
test substance daily throughout the appropriate
treatment period. When given by gavage, the dose
may be based on the weight of the females at the
start of substance administration, or,
alternatively, in view of the rapid weight gain
which takes place during pregnancy, the animals
may be weighed periodically and the dosage based on
the most recent weight determination.
G. Observation of animals
‘1. A gross examination should be made at least
once each day.
2. Additional observations should be made daily
with appropriate actions taken to minimize
loss of animals to the study (e.g. , necropsy
or refrigeration of those animals found dead
and isolation or sacrifice of weak or
moribund animals).
3. Signs of toxicity should be recorded as they
are observed, including the time of onset,
the degree and duration.
4. During the treatment and observation periods,
cage—side observations should include, but
not be limited to: changes in skin and fur,
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October 1984
eye and mucous membranes, as well as
respiratory, autonomic and central nervous
systems, somatomotor activity and behavioral
pattern.
5. Measurements should be made weekly of food
consumption for all animals in the study.
6. Animals should be weighed at least weekly.
7. Females showing signs of abortion or
premature delivery should be sacrificed and
subjected to a thorough macroscopic
examination.
3. Gross necrop y
1. At the time of sacrifice or death during the
study, the dam should be examined
macroscopically for any structural
abnormalities or pathological changes which
may have influenced the pregnancy.
2. Immediately after sacrifice or death, the
uterus should be removed, weighed and the
contents examined for embryonic or fetal
deaths and the number of viable fetuses. The
degree of resorption should he described in
order to help estimate the relative time of
death.
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October 1984
3. The number of corpora lutea should be
determined for all species except mice.
4. The sex of the fetuses should be determined
and they should be weighed individually, the
weights recorded, and the mean fetal weight
per litter derived.
5. Following removal, each fetus should be
examined externally.
6. For rats, mice and hamsters, one—third to
one—half of each litter should be prepared
and examined for skeletal anomalies, and the
remaining part of each litter should be
examined for soft tissue anomalies using
appropriate methods.
7. For rabbits, each fetus should be examined by
careful dissection for visceral anomalies and
then examined for skeletal anomalies.
VI. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form, showing
for each test group: the number of animals at the
start of the test, the number of pregnant animals,
the number and percentages of live fetuses per
litter and the number of fetuses per litter with
any soft tissue or skeletal abnormalities.
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B. Evaluation of results
The findings of a developmental toxicity study
should be evaluated in terms of the observed
effects and the exposure levels producing
effects. It is necessary to consider the
historical developmental toxicity data on the
species/strain tested. A properly conducted
developmental toxicity study should provide a
satisfactory estimation of a no—effect level.
C. Test report
In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice
Standards [ Subpart J, Part 792, Chapter I of Title
40. Code of Federal Regulations] the following
specific information should be reported:
1. Toxic response data by dose;
2. Species and strain;
3. Time of death during the study or
whether animals survived to termination;
4. Time of onset and duration of each
abnormal sign and its subsequent course;
5. Food, body weight, weight gain and uterine
weight data;
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October 1984
6. Pregnancy and litter data; and
7. Fetal data (live/dead, sex, soft tissue
and skeletal defects, resorptions).
VII. REFERENCES
The following references may be helpful in developing
acceptable procotols, and provide a.background of
information on which this section is based. They should
not be considered the only source of information on test
performance, however.
1. H alth Protection Branch. 1975. Ministry of
Health and Welfare. The Testing of Chemicals for
Carcinogenicity, Mutagenicity andTeratogenicity.
Canada: The Honorable Marc Lalonde, Minister of
Health and Welfare, Ministry of Health and
I Welfare. 183 pp.
2. NAS. 1977. National Academy of Sciences.
Principles and Procedures for. Evaluating the
Toxicity of Household Substances. Washington,
D.C.: A report prepared by the Committee for the
Revision of NAS Publication 1138, under the
auspices of the Committee on Toxic9logy, National
Research Council, National Academy of Sciences.
130 pp.
42
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3. WHO. 1967. World Health Organization. Principles
for the Testing of Drugs for Teratogeriicity. WHO
Technical Report Series No. 364. Geneva: World
Health OrganIzation. 18 pp.
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October 1984
INHALATION DEVELOPMENTAL TOXICITY STUDY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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October 1984
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of an inhalable material such as a gas,
volatile substance, or aerosol/particulate, determination
of the potential developmental toxicity is important. The
inhalation developmental toxicity study is designed to
provide information on the potential hazard to the unborn
which may arise from exposure of the mother during
pregnancy.
II. DEFINITIONS
A. Developmental toxicity is the induction of adverse
effects on the developing organism as a result of
in utero exposure to an agent. It is a generic
term which includes endpoints such as resorptions,
structural abnormalities, growth retardation as
well as functional and behavioral deficits.
B. “Aerodynamic diameter” is the diameter
of a sphere of unit density which behaves
aerodynamically like the particles of the test
substance. It is used to compare particles of
different sizes, shapes and densities and to
predict where in the respiratory tract such
particles may be deposited. This term is used in
contrast to “optical,” “measured” or “geometric”
diameters which are representation of actual
diameters and in themselves cannot be related to
deposition within the respiratory tract.
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C. Geometric mean diameter” or “median diameter” is
the calculated aerodynamic diameter which divides
the particles of an aerosol in half based on the
weight of the particles. Fifty percent of the
partic1e by weight will be larger than the median
diameter and 50 percent of the particles will be
smaller than the median diameter. The median
diameter and its geometric standard deviation are
used to statistically describe the particle size
distribution of any aerosol based on the weight and
size of the particles.
D. lnhalable diameter” refers to that aerodynamic
diameter of a particle which is considered to be
inhalable by an organism. Particles which are
capable of being inhaled may be deposited anywhere
within the respiratory tract from the trachea to
the deep lung (the alveoli).
E. Concentration refers to exposure level.
Exposure is expressed as weight or volume of test
substance per volume of air (mg/l ), or as parts per
million (ppm).
F. No—observed—effect level is the maximum
concentration in a test which produces no
observed adverse effec s. A no—observed—effect
level is expressed in terms of weight or volume of
test substance given daily per unit volume of air
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UG—Organ/Tissue—Dev Tox—Inhal
October 1984
III. PRINCIPLE OF THE TEST METHOD
The test substance is administered in graduated
concentrations, for at least that part of the pregnancy
covering the major period of organogenesis, to several
groups of pregnant experimental animals, one exposure
level being used per group. Shortly before the expected
date of delivery, the pregnant females are sacrificed, the
uteri removed, and the contents examined for embryonic or
fetal deaths, and live fetuses.
IV. LIMIT TEST
If a test at an exposure of 5 mg/l (actual concentration
of inhalable substances) or, where this is not possible
due to physical or chemical properties of the test
substance, the maximum attainable concentration, produces
no observable developmental toxicity, then a full study
using three exposure levels might not be necessary.
V. TEST PROCEDURES
A. Animal selection
1. Species and strain
Testing should be performed in at least 2
mammalian species. Commonly used species
include the rat, mouse, rabbit, and
hamster. If other mammalian species are
used, the tester should provide
justification/reasoning for their
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selection. Commonly used laboratory strains
should be employed. The strain should not
have low fecundity and should preferably be
characterized for its sensitivity to
developmental toxins.
2. Age
Young adult animals (nulliparous females)
should be used.
3. Sex
Pregnant female animals should be used at
each exposure level.
4. Number of animals
At least 20 pregnant rats, mice or hamsters
or 12 pregnant rabbits are recommended at
each exposure level. The objective is to
ensure that sufficient pups are produced to
permit meaningful evaluation of the potential
developmental toxicity of the test substance.
B. Control group
A concurrent control group should be used. This
group should be exposed to clean, filtered air
under conditions identical to those used for the
group exposed to the substance of Interest. In
addition, a vehicle—exposed group may be necessary
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October 1984
when the substance under study requires a vehicle
for delivery. It is recommended that during
preliminary range finding studies, air vs. vehicle
exposure be compared. If there is no substantial
difference, air exposure itself would be an
appropriate control. If vehicle and air exposure
yield different results, both vehicle and air
exposed control groups are recommended.
C. Dose levels and dose selection
1.. At least 3 dose levels with a control and,
where appropriate, a vehicle control, should be
used.
2. The vehicle should neither be developmentally
toxic nor have effects on reproduction.
3. To select the appropriate dose levels, a
pilot or trial study may be advisable. Since
pregnant animals have an increased minute
ventilation compared to non—pregnant animals,
It is recommended that the trial study be
conducted in pregnant animals. Similarly,
since presumeably the minute ventilation will
vary with progression of pregnancy, the
animals should be exposed during the same
period of gestation as in the main study. In
the trial study, the concentration producing
embryonic or fetal lethalities or maternal
toxicity should be determined.
49
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October 1984
4. Unless limited by the physical/chemical
nature or biological properties of the
substance, the highest concentration level
should induce some overt maternal toxicity
such as weight loss or decrease in weight
gain, but not more than 10 percent maternal
deaths.
5. The lowest concentration level should not
produce any grossly observable evidence of
either maternal or developmental toxicity.
6. Ideally, the intermediate
c ncentration level(s) should produce minimal
observable toxic effects. If more than one
intermediate concentration is used, the
concentration levels should be spaced to
produce a gradation of toxic effects.
D. Exposure duration
The duration of exposure should be at least six
hours daily allowing appropriate additional time
for chamber equilibrium.
E. Observation period
Day 0 in the test is the day on which a vaginal
plug and/or sperm are observed. The exposure
period should cover the period of major
organogenesis. This may be taken as days 6—15 for
rat and mouse, 6—14 for hamster, or 6—18 for
rabbit.
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Tox—Inhal
October 1984
F. Inhalation exposure
1. The animals Should be tested in inhalation
equipment designed to sustain a dynamic air
flow of 12 to 15 air changes per hour,
ensure an adequate oxygen content of 19
percent, and an evenly distributed exposure
atmosphere. Where a chamber is used, its
design Should minimize crowding of the test
animals and maximize their exposure to the
test substance. This is best accomplished by
individual caging. As a general rule, to
ensure stability of a chamber atmosphere, the
total “volume” of the test animals Should not
exceed 5 percent of the volume of the test
chamber.-
Pregnant animals should be subjected to the
minimum amount of stress. Since whole—body
exposure appears to be the least
stressful mode of exposure it is the
preferred method. In general, oro—nasal or
head—only exposure, which is sometimes used
to avoid excessive concurrent exposure by the
derma]. or oral routes, is not recommended
because of the associated stress accompanying
the restraining of the animals. However,
there may be specific instances where it may
be more appropriate than whole—body
exposure. The tester Should provide
iustification/reasonjng for its selection.
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October 1984
2. A dynamic inhalation system with a suitable
concentration control/monitoring system
should be used. The system should
be adjusted to ensure that conditions
throughout the exposure chamber are
essentially the same. Maintenance of slight
negative pressure inside the chamber will
prevent leakage of the test substance into-
the surrounding area.
3. The temperature at which. the test is
performed should be maintained at 22° C (4-
2°) for rodents or 200 C (+3°) for rabbits,
Ideally, the relative humidity should be
maintained between 40 to 60 percent, but in
certain instances (e.g. tests of aerosols,
use of water vehicle), this may not be
practicable.
G. Physical measurements
Measurements or monitoring of the following
should be conducted:
1., The rate of air flow shpuld be monitored
continuously and- should be recorded at least
every 30 minutes.
2. The actual concentrations of the test
substance should be’ measured in the breathing
zone. During the exposure period the actual
concentrations of the test substance should
52
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be held as constant as practicable, monitored
continuously and recorded at least at the
beginning, at an intermediate time and at the
end of the exposure period.
3. The behavior of the particle generating
system should be established for each
substance under study. In effect, particle
size analysis should be performed to
establish the stability of the particle size
distribution of the aerosol. During
exposure, analysis should be conducted as
often as necessary to determine the
consistency of the particle size
distribution. Likewise, the particle
concentration in the atmosphere should be
established and monitored.
4. Temperature and humidity should be monitored
continuously and recorded at least
every 30 minutes.
H. Food and water during exposure period
Food should be withheld during exposure. Water may
or may not be withheld. If it is not withheld it
should not come in direct contact with the test
atmospheres.
I. Observation of animals
1. A gross examination should be made at least
once each day.
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October 1984
2. Additional observations should be made daily
with appropriate actions taken to minimize
loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead
and isolation or sacrifice of weak or
moribund animals).
3. 3igns of toxicity should be recorded as they
are observed, including the time of onset,
the degree and duration.
4. During the treatment and observation periods,
cage—side observations should include, but
not be limited to: changes in skin and fur,
eye and mucous membranes, as well as
respiratory, autonomic and central nervous
systems, somatomotor activity and behavioral
pattern. Particular attention should be
directed to observation of tremors,
convulsions, salivation, diarrhea, lethargy,
sleep and coma.
5. Measurements should be made weekly of food
consumption for all animals in the study.
6. Animals should be weighed at least weekly.
7. Females showing signs of abortion or
premature delivery should be sacrificed and
subjected to a thorough macroscopic
examination.
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October 1984
J. Gross necropsy
1. At the time of sacrifice or death during the
study, the dam should be examined
rnacroscopically for any structural
abnormalities or pathological changes which
may have influenced the pregnancy.
2. Immediately after sacrifice or death, the
uterus should be removed, weighed and the
contents examined for embryonic or fetal
deaths and the number of viable fetuses. The
degree of resorption should be described in
order to help estimate the relative time of
death.
3. The number of corpora lutea should be
determined for all species except mice.
4. The sex of the fetuses should be determined
and they should be weighed individually, the
weights recorded, and the mean fetal weight
per litter derived.
5. Following removal, each fetus should be
examined externally.
6. For rats, mice and hamsters, one—third to
one—half of each litter should be prepared
and examined for skeletal anomalies, and the
remaining part of each litter should be
examined for soft tissue anomalies using
appropriate methods.
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October 1984
7. For rabbits, each fetus should be examined by
careful dissection for visceral anomalies and
then examined for skeletal anomalies.
VI. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form, showing
for each test group: the number of animals at the
start of the test, the number of pregnant animals,
the number and percentages of live fetuses per
litter and the number of fetuses per litter with
any soft tissue or skeletal abnormalities.
B. Evaluation of results
The findings of a developmental toxicity study
should he evaluated in terms of the observed
effects and the exposure levels producing
effects. It is necessary to consider the
historical developmental toxicity data on the
species/strain tested. A properly conducted
developmental toxicity study should provide a
satisfactory estimation of a no—effect level.
C. Test report
In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice
Standards [ Subpart J, Pa t 792, Chapter I of Title
40. Code of Federal Regulations] the following
specific information should be reported:
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October 1984
1. Test conditions
(a) Description of the exposure apparatus
including design, type, dimensions, source of
air, system for generating particulates/
aerosols, gas or volatile substance, methods
of conditioning air, and the method of
housing the animals in the chamber.
(b) Description of the equipment for
measuring temperature, humidity, and
particulate/aerosol concentrations and size
or concentration of the gas or volatile
substance.
2. Exposure data
The following data shall be tabulated and
presented with mean values and a measure of
variability (eg., standard deviation):
a. Airflow rate through the test chamber;
b. Temperature of air;
c. Nominal concentration——total amount of
test substance fed into the inhalation
equipment divided by volume of air (no
standard deviation);
5k . .I
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October 1984
d. Measured total concentration of
particulates/aerosols/gaseous phases in the
test atmosphere at breathing zone level; and
e. Particle size distribution (e.g., median
aerodynamic diameter of particles with
geometric standard deviation) including
estimate of the percentages of the
inhalable and non—inhalable portions for the
test animals.
3. Animal data
a. Toxic response data by dose;
b. Species and strain;
c. Time of death during:the study or
whether animals survived to termination;
d. Time of onset and duration of each
abnormal sign and its subsequent course;
e. Food, body weight, weight gain and
uterine weight data;
f. Pregnancy and litter data; and
g. Fetal data (live/dead, sex, soft tissue
and skeletal defects, resorptions).
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VII. REFERENCES
The following references may be helpful in developing
acceptable procotols, and provide a background of
information on which this section is based. They should
not be considered the only source of information on test
performance, however.
1. Cage JC. 1970. Experimental inhalation toxicology.
In: Methods in toxicology. Paget GE, ed.
Philadelphia: F.A. Davis Company, pp. 258—277.
2. Health Protection Branch. 1975. Ministry of
Health and Welfare. The Testing of Chemicals for
Carcinogenicity, Mutagenicity and Teratogenicity.
Canada: The Honorable Marc Lalonde, Minister of
Health and Welfare, Ministry of Health and
Welfare. 183 pp.
3. MacFarland HN. 1976. Respiratory toxicology. In:
Essays in toxicology, Vol. 7. Hayes WJ, ed. New
York: Academic Press, pp. 121—154.
4. WAS. 1977. National Academy of Sciences.
Principles and Procedures for Evaluating the
Toxicity of Household Substances. Washington,
D.C.: A report prepared by the Committee for the
Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicoloqy, National
Research Council, National Academy of Sciences.
130 pp.
‘7,
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October 1984
5. WHO. 1967. World Health Organization. Principles
for the Testing of Drugs for Teratogenicity. WHO
Technical Report Series No. 364. Geneva: World
Health Organization. 18 pp.
w
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October, 1984
PRELIMINARY DEVELOPMENTAL TOXICITY SCREEN
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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October, 1984
I. PURPOSE
The In vivo developmental toxicity assay is designed to
assess the potential of agents to induce toxic effects in the
conceptus after administration to the mother during pregnancy.
This test should be used only to prioritize environmental agents
for testing by more rigorous standard protocols (see TSCA
Guidelinees Developmental Toxicity Study).
II. DEFINITIONS
A. Developmental toxicity is the induction of adverse
effects on the developing organism as a result of in
utero exposure to an agent. It is a generic term which
includes endpoints such as resorption, structural
abnormalities, growth retardation, as well as
functional deficits.
B. Dose is the amount of test substance administered, and
is expressed as weight of test substance (g, mg) per
unit weight of a test animal (e.g. mg/kg).
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October, 1984
C. No—observed—effect level is the maximum concentration
in a test which produces no observed adverse effects.
A no—observed—effect level is expressed in terms of
weight of test substance given daily per unit weight of
test animal (mg/kg).
III. PRZNCIPL,E OF THE TEST METHOD
The test substance is administered to pregnant animals
during a significant portion of the period of major
organogenesis. A single dose level is administered. This dose
level should be high enough to elicit overt maternal toxicity.
This toxicity should not exceed a mortality level of 10% or a
reduction of overall maternal weight gain of more than 30% of the
control value during the treatment period. The dams are allowed
to give birth and the neonates are counted and weighed on days 1
and 3 postpartum (day 1 is the day of birth). The underlying
hypothesis for this assay is that a wide spectrum of
developmentally toxic effects will be expressed as intrauterine
death or impaired neonatal growth or survival.
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October, 1984
IV. TEST PROCEDURES
A. Animal selection
1. Species and strain
Testing must be performed in a mammalian species
and strain which will allow human handling of
newborn pups without cannibalization or
ab ndonment. The preferred species is either rat
or mouse. The strain should be commonly used and
should not have low fecundity.
2. Age
Young adult animals should be used,.
3. Sex
Pregnant (primigravid) female animals should be
used.
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October, 1984
4. Number of animals
Sufficient animals should be used to produce a
sample size of at least 20 pregnant animals at
term. The objective is to ensure that sufficient
litters are produced to permit meaningful
evaluation of the potential developmental toxicity
of the test substance.
B. Control group
A concurrent control group large enough to produce at
least 30 pregnant females at term should be used. This
group should be an untreated or sham treated control
group, or, if a vehicle is used in administering the
test substance, a vehicle control group. Except for
treatment with the test substance, animals in the
control group(s) should be handled in an identical
manner to test group animals.
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October, 1984
C. Dose le ,e1s and dose selection
1. A single dose level with a concurrent control
should be used. It may be advisable to use a
second dose level which is some fraction. (e.g.,
30—50%) of the first dose level. This will
facilitate the ‘identification of studies with
potentially false positive results.
2. The vehicle should be neither developmentally
toxic nor have effects on reproduction in the
dams. If there is uncertainty asto the
developmental toxicity potential of the vehicle, a
sham control group should be used also.
3. To select the appropriate dose levels, a pilot or
trial study should be conducted. If a pilot study
is not done, a rationale or justification should
be provided.
4. Unless limited by the physical/chemical nature or
biological properties of the substance, the dose
level used should be:
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October, 1984
(a) high enough to cause overt maternal toxicity
as evidenced by death, significant weight
loss or reduced weight gain, or
(b) 1000 mg/kg, if lower dose levels fail to
induce maternal toxicity.
D. Observation period
Day 0 in the test is the day in which a vaginal plug
and/or sperm are observed. The dose period should
encompass a significant portion of the period of major
organogenesis. This may be taken as days 6—15 for the
rat and mouse.
E. Administration of test substance
The test substance or vehicle is usually administered
orally, by intubation unless the chemical or physical
characteristics of the test substance or pattern of
human exposure suggest a more appropriate route of
administration. The test substance should be
administered at the same time each day.
F. ! posure conditions
Female test animals should be housed individually and
provided with bedding. They should be treated with the
test substance daily throughout the appropriate
treatment period. When given by gavage, in view of the
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October, 1984
rapid weight gain which takes place during
pregancy, the animals should be weighed frequently and
the dosage based on the most recent weight
determination.
G. Observation of pregnant animals
1. Pregnant animals should be observed at least once
daily throughout the study, or until they die or
are sacrificed.
2. During the treatment and observation periods,
cage—side observations should include, but not be
limited to: changes in skin and fur, eye and
mucous membranes, as well as respiratory,
autonomic and central nervous systems, somatomotor
activity and behavioral pattern.
3. Signs of toxicity should be recorded as they are
observed, including the time of onset, the degree
and duration.
4. During the dosing period females that die or are
sacrificed because they are moribund should be
exmained for dosing errors and signs of pregnancy;
details of the conditions of the uterus and/or its
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October, 1984
contents should be recorded. Animals that have
not delivered three days after the expected date
of parturition should be sacrificed and similar
examinations made. Uteri that do not show gross
evidence of pregnancy may be chemically treated
(e.g. 10% ammonium sulfide) to improve detection
of early termination of pregnancy.
H. Observation of dams after birth
Dams should be observed for signs of overt toxicity
during the postpartum period at the same time neonatal
examinations are being made.
I. Neonatal examinations
1. Dams are allowed to give birth and the litters are
examined for gross anomalies and presence of milk,
counted, and weighed on postpartum days 1 and 3.
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HG—Organ/Tissue—Dev Tox Screen
October, 1984
2. Dead pups may be preserved and subsequently
necropsied and abnormalities noted.
3. For those compounds that induce only neonatal
growth reduction, litters should be normalized on
postpartum day 3 (to approximately four females
and four males) and left with the dam through
weaning. This procedure will determine if the
growth reduction is transient or if it represents
a permanent functional alteration.
V. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form, showing for
each test group: the number of animals at the start of
the test, the number of pregnant animals, the average
duration of pregnancy, the maternal weight gain during
the treatment period, the number of dams with a viable
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HG—Organ/Tissue—Dev Tox Screen
October, 1984
litter (at least one live neonate), the average number
of live neonates per litter on days 1 and 3, the
average neonatal weight on days 1 and 3, the average
neonatal weight gained during that period, and data on
necropsied pups.
B. Evaluation of results
The findings of this bioassay should be evaluated in
terms of the types of effects noted. All data analyses
should compare treatment groups with their concurrent
controls. Statistical treatment of the results should
involve analysis of variance, and the number of live
pups on days 1 and 3 should be used as a covariate in
the analyses of postnatal body weight so as to correct
for differences in pup weights due to litter size.
Fully resorbed females should be considered to have
zero line on days 1 and 3. The number of animals going
to term must be sufficiently large to allow for a
reasonable detection of compound—induced deficiences.
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HG—Organ/Tissue—Dev Tox Screen
October, 1984
Conditions which significantly reduce the number of
dams going to term (e.g. lack of pregn’ancy or compound—
induced materna1 death) should lead to a repeat of the
study. Lack of both overt maternal toxicity and
reproductive effects also should lead to a repeat of
the study if the dose level used was less than 1000
mg 1kg.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards (Subpart
3, Part 792, Chapter I of Title 40. Code of Federal
Regulations) the following specific information should
be reported:
1. Toxic response data;
2. Species and strain;
3. Time of maternal death during the studyor whether
animals survived to termination;
4. Time of onset and duration of each abnormal sign
and its subsequent course;
5. Pregnancy data;
-,v)
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HG—Organ/Tiasue—Dev Tox Screen
October, 1984
6. Litter data including number live and dead; and
average litter weight on days 1 and 3 postpartum;
and
7. Necropsy data on dead pups, if such were
generated.
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II(’ .—Organ/Tissue—Dev Tox Screen
Uctoher, 1984
Vt • ____________
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Chernoff N and Kaviock R. 1982. An in vivo teratology
screen utilizing pregnant mice. 1. Toxicol. Environ.
Health 10: 541—550.
2. Doe JE, Samuels DM, Tiriston DJ and De Silva
Wickramaratne GA. 1983. Comparative aspects of the
reproductive toxicology by inhalation in rats of
ethylene glycol monomethyl ether and propylene glycol
monomethyl ether. Toxicol. Appi. Pharmacol. 69(1): 43—
47.
14 1)’!
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HG—Gone Muta—S. typhimurium
October, 1984
THE SAUIONELLA TYPHIMURIUM REVERSE
MUTATION ASSAY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF’ PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
p - i ,-
I 1.)
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I(;—(;(sr’ ’ Miii d . typh i ITIII r t iiui
Oc Lobe r , 9H 4
I. PURPOSE
The Salmonella typhimurium histidine (his) rever ion system
is a microbial assay which measures hi >hls reversion
induced by chemicals which cause base changes or frameshift
mutations in the genome of this organism.
II. DEFINITIONS
A. A reverse-mutation assay in Salmonefla typhimurium
detects mutation in a gene of a hist din requirinq
strain to produce a histidine independent strain of Lhi ;
organism.
B. Base pair mutagens are agents which cause a base change
in the DNA. This change may occur at the site of the
original mutation or at a second site in the DNA
molecule. -
C. Frameshift mutagens are agents which cause the addition
or deletion of single or multiple base pairs in the DNA
molecule.
III. REFERENCE SUBSTANCES
These may include, but need not he limited to, sodium azide,
nitroturantoi n, 4—nitro—o—phenylene—diami ne and 2—
aminoanthracene for the plate incorporation method and
direct blue 6 for the azo—reduction method.
IV. TEST METHOD
A. Principle
Bacteria are exposed to test chemical with and without a
metabol ic activation system and plated onto minimal
medium. After a suitable period of incubation,
revertant colonies are counted a nd compared to the
number of spontaneous revertants in an untreated and/or
vehicle control plates.
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JIG—Gene Muta—S. typhitnurium
October, 1984
B. Description
Several methods for performing the test have been
described. Among those used are:
1. the direct plate incorporation method,
2. the azo—reductjon method,
3. the preincuhatjon method,
4. the dessicator method, and
5. the suspension method
The procedures described here are for the direct plate
incorporation method and the azo—reduction method.
C. Strain selection
1. Designation
At the present time four strains, TA 1535, TA 1537,
TA 98 and TA 100 should he used. The use of other
strains in addition to these four is left to the
discretion of the investigator.
2. Preparation and storage
Scientifically accepted methods of stock culture
preparation and storage should he used. The
requirement of histidine for growth should be
demonstrated for each strain. Other phenotypic
characteristics should he checked using such
methods as crystal violet sensitivity and
resistance to ampicillin. The extent of
spontaneous mutation should be in the range
expected either as reported in the literature or as
established in the laboratory by historical control
values.
3. Bacterial growth
Fresh cultures of bacteria should he qrown up to
the late exponential or arl stationary ph t t e of
growth (approximately 10 —10 cells per nil).
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HG—Gene Muta—S. typhimurium
October, 1984
D. Metabolic activation
Bacteria should be exposed to the test substance both in
the presence and absence of an appropriate metabolic
activation system. For the direct plate incorporation
method, the most commonly used system is a c factor
supplemented postmitochondrial fraction prepared from
the livery of rodents treated with enzyme inducing
agents such as Aroclor 1254. For the azo—reductiton
method, a cofactor supplemented postmitochondrial
fraction prepared from the livers of untreated hamsters
‘is preferred. For this method, the cofactor supplement
should contain flavin mononucleotide, exogenous glucose—
6—phosphate dehydrogenase, NADH and excess of glucose-6—
phosphate.
E. Control groups
1. Concurrent controls
Concurrent positive and negative (untreated and/or
vehicle) controls should he included in each
experiment. Positive controls should insure both
strain responsiveness and efficacy of the metabolic
activation system.
2. Strain specific positive controls
Strain specific positive controls should be
included in the assay. Examples of such controls
are as follows:
a. Strain TA 1535, sodium azide;
b. Strain TA 100, nitroturantoin
c. TA 98, TA 1537, 4—nitro—o—phenylene—diamine
3. Positive controls to ensure the efficacy
of the activation system
The positive control reference substance br test-s
including a metabolic activation system should he
s elected on the basis of the type of activation
system used in the test. 2—Aminoanthracene is an
example of a positive control compound in plate—
incorporation tests using postmitochondrial
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If(;—Gene Muta—S. typhimurium
October, 1984
fractions from the livers of rodents treated with
enzyme inducing agents such as Aroclor—l254. Direct
Blue 6 is an example of a positive control compound
in the azo—reduction method. Other positive
control reference substances may be used.
4. Class—specific positive controls
The azo—reduction method should include positive
controls from the same class of compounds as the
test agent wherever possible. For example,
benzidine based dyes with known mutagenic potential
should be used as controls in experiments with
henzidine based dyes of unknown mutagenic
potential.
F. Test chemicals
1. Vehicle
Test chemicals and positive control reference
substances should be dissolved in an appropriate
vehicle and then further diluted in vehicle for use
in the assay.
2. Exposure concentrations
a. The test should initially be performed over a
broad rarhge of concentrations. Among the
criteria to be taken into consideration for
determining the upper limits of test chemical
concentration are cytotoxicity and
solubility. Cytotoxicity of the test chemical
may be altered in the presence of metabolic
activation systems. Toxicity may he evidenced
by a reduction in the number of spontaneous
revertants, a clearing of the background lawn
or by the appearance of pinpoint norirevertant
colonies. Relatively insoluble compounds
should be tested up to the limits of
solubility. For freely soluble nontoxic
chemicals, the upper test chemical
concentration should be determined on a case
by case basis.
b. Generally, a maximum of 5 mg/plate for pure
substances is considered acceptable. At least
5 different amounts of test substance should
be tested with adequate intervals between test
points.
V ‘1
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UG—Gene Muta—S. typhimurium
October, 1984
c. A suspected positive response not showing a
clear dose—related response should be
confirmed by testing over a narrow range of
concentrations.
V. TEST PERFORMANCE
A. Direct plate incorporation method
For this test without met’aholic activation, test
chemical and 0.1 ml of a fresh bacterial culture should
be added to 2.0 ml of overlay agar. For tests with
metabolic activation, 0.5 ml of activation mixture
containing an adequate amount of postmitochondrial
fraction should be added to the agar overlay after the
addition of test chemical and bacteria. Contents of
each tube !should be mixed and poured over the surface f
a selecti e agar plate. Overlay agar should be allowed
to solidify before incubation. At the end of the
incubation period, revertant colonies per plate should
be counted.
B. Azo—reduction method
For this test, 0.5 S—9 mix containing 150 ul of S—9 and
0.1 ml of bacterial culture should he added to a test
tube kept on ice. Chemical should be added and the
tubes should be incubated without shaking at 30 C for 30
mm. At the end of the incubation period, 2.0 ml of
agar should be added to each tube, the contents mixed
and poured over the surface of a selective agar plate.
Overlay agar should be allowed to solidify before
incubation. At the end of the incubation period,
revertant colonies per plate should be counted.
It is recommended that all azo dyes be tested in either
the plate incorporation or preincubation method and in
the azo-reduction method.
C. Other methods
Other methods may also be appropriate.
D. Media
An appropriate selective medium with an adequate overlay
agar should be used.
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HG—Gene Muta—S. typhimurium
October, 1984
E. Incubation conditions
All plates within a given exp ?rIInont 3hot11d be IncuhaLed
for the same time period. This incuhation period shoull
be for 48—72 hours at 37 C.
F. Number of cultures
In general, all plating should be done at least in
triplicate. If scientifically justified, the use of
duplicates may he acceptable. All results should be
confirmed in an independent experiment.
VI. DATA AND REPORT
A. Treatment of results
Data should be presented as number of revertant colonies
per plate for each replicate and dose. The numbers of
revertant colonies on both negative (untreated and/or
vehicle) and positive control plates should also be
presented. Individual plate counts, the mean number of
revertant colonies per plate and standard deviation
should be presented for test chemical and positive and
negative (untreated and/or vehicle) controls.
B. Statistical evaluation
Data should be evaluated by appropriate statistical
methods.
C. Interpretation of results
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose—related increase in the number of
revertants. Another criterion may be based upon
detection of a reproducible and statistically
significant positive response for at least one of
the test substance concentrations.
2. A test substance which does not produce either a
statistically significant dose—related increase in
the number of revertants or a statistically
significant and reproducible positive response at
any one of the test points is considered non—
mutagenic in this system.
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HG-Gene Muta—S. typhimurium
October, 1984
3. Both biological and statistical significance should
be considered together in the evaluation.
D. Test evaluation
1. Positive results from the S. typhimurium reverse
mutation assay indicate that, under the test
conditions, the test substance induces point
mutations by base changes or frameshifts in the
genome of this organism. -
2. Negative results indicate that under the test
conditions the test substance is not mutagenic in
S. typhimurium .
E. Test report
In addition to the reporting recommendations as
specified in the EPA Good Laboratory Practices Standards
(Subpart 1, Part 792, Chapter I of Title 40. Code of
Federal Regulations) the following specific information
should be reported:
1. bacterial strains used;
2. metabolic activation system used and its
preparation for use in the assay; for 5—9
preparations this should include cofactor cocktail
(contents, storage conditions and amount used);
source and amount of S—9 and details of preparation
and storage of S—9 mix;
3. dose levels and rationale for selection of dose;
4. positive and negative controls;
5. individual plate counts, mean number of revertarit
colonies per plate, standard deviation;
6. dose—response relationship, if applicable.
82
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lG—Gene Muta—S. typhimurium
October, 1984
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Ames BN, McCann J, Yamasakj E. 1975. Methods for
detecting carcinogens and mutagens with the
Salmonella/mammal jan—microsorne mutagenicity test.
Mutation Research 31:347—364.
2. de Serres FJ, Shelby MD. 1979. The Salmonella
mutagenicity assay: recommendations. Science 203:563—
565.
3. Prival MJ, Bell SJ, Mitchell VD, Peiperl MD, Vaughn VL.
1984. Mutagenicity of benzidine and henzidine—congener
dyes and selected monoazo dyes in a modified Salmonella
assay. Mutation Res. 136:33—47.
4. Prival MJ, Mitchell VD. 1982. Analysis of a method for
testing azo dyes for mutagenic activity in Salmonella
yphimurium in the presence of flavin mononculeotide and
hamster liver S—9. Mutation Res 97:103—116.
5. Vogel HJ, Bonner DM. 1956. Acetylornithjnase of E.
coli : partial purification and some properties. J Bioj.
Chem 218:97—106.
6. Yahagi T, Degawa M, Seino Y, Matsushima T, Nagao M,
Sugimura T, Hashimoto Y. 1975. Mutagenicity of
carcinogenic azo dyes and their derivatives. Cancer
Letters 10:91—96.
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