United States Prevention, Pesticides EPA 712-C-98-211
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.4200
Carcinogenicity
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. 136, etseq. ).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 870.4200 Carcinogenicity.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 798.3300 Oncogenicity; OPP
83-2 Carcinogenicity—Two Species, Rat and Mouse Preferred (Pesticide
Assessment Guidelines, Subdivision F—Hazard Evaluation; Human and
Domestic Animals) EPA report 540/09-82-025, 1982; and OECD 451
Carcinogenicity Studies.
(b) Purpose. The objective of a long-term Carcinogenicity study is
to observe test animals for a major portion of their life span for develop-
ment of neoplastic lesions during or after exposure to various doses of
a test substance by an appropriate route of administration.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this guide-
line. The following definitions also apply to this guideline.
Carcinogenicity is the development of neoplastic lesions as a result
of the repeated daily exposure of experimental animals to a chemical by
the oral, dermal, or inhalation routes of exposure.
Cumulative toxicity is the adverse effects of repeated dose occurring
as a result of prolonged action on, or increased concentration of, the ad-
ministered test substance or its metabolites in susceptible tissues.
Dose in a Carcinogenicity study is the amount of test substance ad-
ministered via the oral, dermal or inhalation routes for a period of up to
24 months. Dose is expressed as weight of the test substance (grams, milli-
grams) per unit body weight of test animal (milligram per kilogram), or
as weight of the test substance in parts per million (ppm) in food or drink-
ing water. When exposed via inhalation, dose is expressed as weight of
the test substance per unit volume of air (milligrams per liter) or as parts
per million.
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance.
(d) Test procedures—(1) Animal selection—(i) Species and strain.
Testing should be performed on two mammalian species. Rats and mice
are the species of choice because of their relatively short life spans, limited
cost of maintenance, widespread use in pharmacological and toxicological
studies, susceptibility to tumor induction, and the availability of inbred
or sufficiently characterized strains. Commonly used laboratory strains
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should be used. If other mammalian species are used, the tester should
provide justification/reasoning for their selection.
(ii) Age/weight. (A) Testing should be started with young healthy
animals as soon as possible after weaning and acclimatization.
(B) Dosing should generally begin no later than 8 weeks of age.
(C) At commencement of the study, the weight variation of animals
used should be within 20 percent of the mean weight for each sex.
(D) Studies using prenatal or neonatal animals may be recommended
under special conditions.
(iii) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level.
(B) Females should be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 100 rodents (50 males and 50 females)
should be used at each dose level and concurrent control group.
(B) If interim sacrifices are planned, the number should be increased
by the number of animals scheduled to be sacrificed during the course
of the study.
(C) For a meaningful and valid statistical evaluation of long term
exposure and for a valid interpretation of negative results, the number of
animals in any group should not fall below 50 percent at 15 months in
mice and 18 months in rats. Survival in any group should not fall below
25 percent at 18 months in mice and 24 months in rats.
(D) The use of adequate randomization procedures for the proper allo-
cation of animals to test and control groups is required to avoid bias.
(E) Each animal should be assigned a unique identification number.
Dead animals, their preserved organs and tissues, and microscopic slides
should be identified by reference to the unique numbers assigned.
(v) Husbandry. (A) Animals may be group-caged by sex, but the
number of animals per cage must not interfere with clear observation of
each animal. The biological properties of the test substance or toxic effects
(e.g., morbidity, excitability) may indicate a need for individual caging.
Animals should be housed individually in dermal studies and during expo-
sure in inhalation studies.
(B) The temperature of the experimental animal rooms should be at
22 + 3 °C.
(C) The relative humidity of the experimental animal rooms should
be 50 + 20 percent.
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(D) Where lighting is artificial, the sequence should be 12 hours light/
12 hours dark.
(E) Control and test animals should be fed from the same batch and
lot. The feed should be analyzed to assure uniform distribution and ade-
quacy of nutritional requirements of the species tested and for impurities
that might influence the outcome of the test. Animals should be fed and
watered ad libitum with food replaced at least weekly.
(F) The study should not be initiated until animals have been allowed
a period of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
period of quarantine. An acclimation period of at least five days is rec-
ommended.
(2) Control and test substances, (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle. If a vehicle or dilu-
ent is needed, it should not elicit toxic effects itself. It is recommended
that wherever possible the use of an aqueous solution be considered first,
followed by consideration of solution in oil, and finally solution in other
vehicles.
(ii) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability. Prior to the initiation
of the study, there should be a characterization of the test substance, in-
cluding the purity of the test compound, and, if possible, the name and
quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into feed
or another vehicle, the period during which the test substance is stable
in such a mixture should be determined prior to the initiation of the study.
Its homogeneity and concentration should be determined prior to the initi-
ation of the study and periodically during the study. Statistically random-
ized samples of the mixture should be analyzed to ensure that proper mix-
ing, formulation, and storage procedures are being followed, and that the
appropriate concentration of the test or control substance is contained in
the mixture.
(3) Control groups. A concurrent control group (50 males and 50
females) is required. This group should be untreated or if a vehicle is
used in administering the test substance, a vehicle control group. If the
toxic properties of the vehicle are not known, both untreated and vehicle
control groups are required.
(4) Dose levels and dose selection, (i) For risk assessment purposes,
at least three dose levels should be used, in addition to the concurrent
control group. Dose levels should be spaced to produce a gradation of
effects. A rationale for the doses selected must be provided.
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(ii) The highest-dose level should elicit signs of toxicity without sub-
stantially altering the normal life span due to effects other than tumors.
The highest dose should be determined based on the findings from a 90-
day study to ensure that the dose used is adequate to asses the carcinogenic
potential of the test substance. Thus, the selection of the highest dose to
be tested is dependent upon changes observed in several toxicological pa-
rameters in subchronic studies. The highest dose tested need not exceed
1,000 mg/kg/day.
(iii) The intermediate dose levels should be spaced to produce a gra-
dation of toxic effects.
(iv) The lowest-dose level should produce no evidence of toxicity.
(v) For skin carcinogenicity studies, when toxicity to the skin is a
determining factor, the highest dose selected should not destroy the func-
tional integrity of the skin, the intermediate doses should be a minimally
irritating dose, and the low dose should be the highest nonirritating dose.
(vi) The criteria for selecting the dose levels for skin carcinogenicity
studies, based on gross and histopathologic dermal lesions, are as follows:
(A) Gross criteria for reaching the high dose:
(7) Erythema (moderate).
(2) Scaling.
(3) Edema (mild).
(4) Alopecia.
(5) Thickening.
(B) Histologic criteria for reaching the high-dose:
(7) Epidermal hyperplasia.
(2) Epidermal hyperkeratosis.
(3) Epidermal parakeratosis.
(4) Adnexal atrophy/hyperplasia.
(5) Fibrosis.
(6) Spongiosis (minimal-mild).
(7) Epidermal edema (minimal-mild).
(8) Dermal edema (minimal-moderate).
Inflammation (moderate).
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(C) Gross criteria for exceeding the high-dose:
(7) Ulcers, fissures.
(2) Exudate/crust (eschar).
nonviable (dead) tissues.
(4) Anything leading to destruction of the functional integrity of the
epidermis (e.g., caking, fissuring, open sores, eschar).
(D) Histologic criteria for exceeding the high-dose:
(7) Crust (interfollicular and follicular).
(2) Microulcer.
(3) Degeneration/necrosis (mild to moderate).
(4) Epidermal edema (moderate to marked).
(5) Dermal edema (marked).
(6) Inflammation (marked).
(5) Administration of the test substance. The three main routes of
administration are oral, dermal, and inhalation. The choice of the route
of administration depends upon the physical and chemical characteristics
of the test substance and the form typifying exposure in humans.
(i) Oral studies. If the test substance is administered by gavage, the
animals are dosed with the test substance on a 7-day per week basis for
a period of at least 18 months for mice and hamsters and 24 months for
rats. However, based primarily on practical considerations, dosing by ga-
vage on a 5-day per week basis is acceptable. If the test substance is
administered in the drinking water or mixed in the diet, then exposure
should be on a 7-day per week basis.
(ii) Dermal studies. (A) The animals should be treated with the test
substance for at least 6 h/day on a 7-day per week basis for a period
of at least 18 months for mice and hamsters and 24 months for rats. How-
ever, based primarily on practical considerations, application on a 5 -day
per week basis is acceptable. Dosing should be conducted at approximately
the same time each day.
(B) Fur should be clipped weekly from the dorsal area of the trunk
of the test animals. Care should be taken to avoid abrading the skin which
could alter its permeability. A minimum of 24 hours should be allowed
for the skin to recover before the next dosing of the animal.
(C) Preparation of test substance. Liquid test substances are generally
used undiluted, except as indicated in paragraph (e)(4)(vi) of this guideline.
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Solids should be pulverized when possible. The substance should be moist-
ened sufficiently with water or, when necessary, with a suitable vehicle
to ensure good contact with the skin. When a vehicle is used, the influence
of the vehicle on toxicity of, and penetration of the skin by, the test sub-
stance should be taken into account.The volume of application should be
kept constant, e.g. less than 100 (iL for the mouse and less than 300 (iL
for the rat. Different concentrations of test solution should be prepared
for different dose levels.
(D) The test substance should be applied uniformly over a shaved
area which is approximately 10 percent of the total body surface area.
In order to dose approximately 10 percent of the body surface, the area
starting at the scapulae (shoulders) to the wing of the ileum (hipbone)
and half way down the flank on each side of the animal should be shaved.
With highly toxic substances, the surface area covered may be less, but
as much of the area as possible should be covered with as thin and uniform
a film as practical.
(E) During the exposure period, the application site should not be
covered when mice or hamsters are the species of choice. For rats, the
test substance may be held in contact with the skin with a porous gauze
dressing and nonirritating tape if necessary. 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. The test sub-
stance may be wiped from the skin after the 6-hour exposure to prevent
ingestion.
(iii) Inhalation studies. (A) The animals should be exposed to the
test substance for 6 h/day on a 7-day per week basis, for a period of at
least 18 months in mice and 24 months in rats. However, based primarily
on practical considerations, exposure for 6 h/day on a 5-day per week
basis is acceptable.
(B) The animals should be tested in dynamic inhalation equipment
designed to sustain a minimum air flow of 10 air changes per hour, an
adequate oxygen content of at least 19 percent, and uniform conditions
throughout the exposure chamber. Maintenance of slight negative pressure
inside the chamber will prevent leakage of the test substance into surround-
ing areas.
(C) The selection of a dynamic inhalation chamber should be appro-
priate for the test substance and test system. Where a whole body chamber
is used to expose animals to an aerosol, individual housing must be used
to minimize crowding of the test animals and maximize their exposure
to the test substance. To ensure stability of a chamber atmosphere, the
total volume occupied by the test animals should not exceed 5 percent
of the volume of the test chamber. It is recommended, but not required,
that nose-only or head-only exposure be used for aerosol studies in order
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to minimize oral exposures due to animals licking compound off their fur.
Heat stress should be minimized.
(D) The temperature at which the test is performed should be main-
tained at 22 + 20 °C. 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.
(E) The rate of air flow should be monitored continuously but re-
corded at least three times during exposure.
(F) Temperature and humidity should be monitored continuously but
should be recorded at least every 30 minutes.
(G) The actual concentrations of the test substance should be meas-
ured in the breathing zone. During the exposure period, the actual con-
centrations of the test substance should be held as constant as practicable,
monitored continuously or intermittently depending on the method of anal-
ysis. Chamber concentrations may be measured using gravimetric or ana-
lytical methods as appropriate. If trial run measurements are reasonably
consistent (±10 percent for liquid aerosol, gas, or vapor; + 20 percent
for dry aerosol), the two measurements should be sufficient. If measure-
ments are not consistent, then three to four measurements should be taken.
Whenever the test substance is a formulation, or it is necessary to formu-
late the test substance with a vehicle for aerosol generation, the analytical
concentration must be reported for the total formulation, and not just for
the active ingredient (AI). If, for example, a formulation contains 10 per-
cent AI and 90 percent inerts, a chamber of analytical limit concentration
of 2 mg/L would consist of 0.2 mg/L of the AI. It is not necessary to
analyze inert ingredients provided the mixture at the animal's breathing
zone is analogous to the formulation; the grounds for this conclusion must
be provided in the study report. If there is some difficulty measuring cham-
ber analytical concentration due to precipitation, nonhomogeneous mix-
tures, volatile components, or other factors, additional analysis of inert
components may be necessary.
(H) During the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol concentra-
tions with respect to particle size. Measurement of aerodynamic particle
size in the animals's breathing zone should be measured during a trial
run. If MMAD values for each exposure level are within 10 percent of
each other, then two measurements during the exposures should be suffi-
cient. If pretest measurements are not within 10 percent of each other,
three to four measurements should be taken. The mass median aero-
dynamic diameter (MMAD) particle size range should be between 1-3
(im. The particle size of hygroscopic materials should be small enough
when dry to assure that the size of the swollen particle will still be within
the 1-3 (im range.
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(I) Feed should be withheld during exposure. Water may also be with-
held during exposure.
(6) Observation period. It is necessary that the duration of the car-
cinogenicity study comprise the majority of the normal life span of the
strain of animals used. This time period should not be less than 24 months
for rats and 18 months for mice, and ordinarily not longer than 30 months
for rats and 24 months for mice. For longer time periods, and where any
other species are used, consultation with the Agency in regard to the dura-
tion of the study is advised.
(7) Observation of animals, (i) Observations should be made at least
twice each day for morbidity and mortality. Appropriate actions should
be taken to minimize loss of animals from the study (e.g., necropsy or
refrigeration of those animals found dead and isolation or sacrifice of weak
or moribund animals).
(ii) A careful clinical examination should be made at least once week-
ly. Observations should be detailed and carefully recorded, preferably
using explicitly defined scales. Observations should include, but not be
limited to, evaluation of skin and fur, eyes and mucous membranes, res-
piratory and circulatory effects, autonomic effects such as salivation,
central nervous system effects, including tremors and convulsions, changes
in the level of activity, gait and posture, reactivity to handling or sensory
stimuli, altered strength and stereotypies or bizarre behavior (e.g., self-
mutilation, walking backwards).
(iii) Body weights should be recorded individually for all animals
once pretreatment, once a week during the first 13 weeks of the study
and at least once every 4 weeks, thereafter, unless signs of clinical toxicity
suggest more frequent weighing to facilitate monitoring of health status.
(iv) Measurements of feed consumption should be determined weekly
during the first 13 weeks of the study and at approximately monthly inter-
vals thereafter unless health status or body weight changes dictate other-
wise. Measurements of water consumption should be determined at the
same intervals if the test substance is administered in the drinking water.
(v) Moribund animals should be removed and sacrificed when noticed
and the time of death should be recorded as precisely as possible. At the
end of the study period, all survivors should be sacrificed.
(8) Clinical pathology. At 12 months, 18 months, and at terminal
sacrifice, a blood smear should be obtained from all animals. A differential
blood count should be performed on blood smears from those animals in
the highest dosage group and the controls from the terminal sacrifice. If
these data, or data from the pathological examination indicate a need, then
the 12- and 18-month blood smears should also be examined. Differential
blood counts should be performed for the next lower groups if there is
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a major discrepancy between the highest group and the controls. If clinical
observations suggest a deterioration in health of the animals during the
study, a differential blood count of the affected animals should be per-
formed.
(9) Gross necropsy, (i) A complete gross examination should be per-
formed on all animals, including those that died during the experiment
or were killed in a moribund condition.
(ii) At least the liver, kidneys, adrenals, testes, epididymides, ovaries,
uterus, spleen, brain and heart should be weighed wet as soon as possible
after dissection to avoid drying. The lungs should be weighed if the test
substance is administered by the inhalation route. The organs should be
weighed from interim sacrifice animals as well as from at least 10 animals
per sex per group at terminal sacrifice.
(iii) The following organs and tissues, or representative samples there-
of, should be preserved in a suitable medium for possible future
histopathological examination.
(A) Digestive system—salivary glands, esophagus, stomach, duode-
num, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder
(when present).
(B) Nervous system—brain (multiple sections, including cerebrum,
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial,
preferably in close proximity to the muscle), spinal cord (three levels, cer-
vical, mid-thoracic and lumbar), eyes (retina, optic nerve).
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/hematopoietic system—aorta, heart, bone marrow
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering
the route of administration and another one distant from the route of ad-
ministration to cover systemic effects), spleen.
(F) Urogenital system—kidneys, urinary bladder, prostate, testes,
epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Other—all gross lesions and masses, skin.
(iv) In inhalation studies, the entire respiratory tract, including nose,
pharynx, larynx, and paranasal sinuses should be examined and preserved.
In dermal studies, skin from treated and adjacent control skin sites should
be examined and preserved.
(v) Inflation of lungs and urinary bladder with a fixative is the optimal
method for preservation of these tissues. The proper inflation and fixation
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of the lungs in inhalation studies is essential for appropriate and valid
histopathological examination.
(vi) Information from clinical pathology, and other in-life data should
be considered before microscopic examination, since they may provide sig-
nificant guidance to the pathologist.
(10) Histopathology. (i) The following histopathology should be per-
formed:
(A) Full histopathology on the organs and tissues listed under para-
graph (d)(9)(iii) of this guideline of all animals in the control and high-
dose groups and all animals that died or were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) If the results show substantial alteration of the animal's normal
life span, the induction of effects that might affect a neoplastic response,
or other effects that might compromise the significance of the data, the
next lower dose levels should be examined as described under paragraph
(d)(10)(i) of this guideline.
(iii) An attempt should be made to correlate gross observations with
microscopic findings.
(iv) Tissues and organs designated for microscopic examination
should be fixed in 10 percent buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours prior
to trimming.
(e) Data and reporting—(1) Treatment of results, (i) 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 animals showing lesions,
the types of lesions, and the percentage of animals displaying each type
of lesion.
(ii) When applicable, all observed results (quantitative and qualitative)
should be evaluated by an appropriate statistical method. Any generally
accepted statistical methods may be used; the statistical methods including
significance criteria should be selected during the design of the study.
(2) Evaluation of study results, (i) The findings of a carcinogenicity
study should be evaluated in conjunction with the findings of previous
studies and considered in terms of the toxic effects, the necropsy and
histopathological findings. The evaluation should include the relationship
between the dose of the test substance and the presence, incidence, and
severity of abnormalities (including behavioral and clinical abnormalities),
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gross lesions, identified target organs, body weight changes, effects on
mortality, and any other general or specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects, fur-
ther investigation to establish absorption and bioavailablity of the test sub-
stance should be considered.
(iii) In order for a negative test to be acceptable, it should meet the
following criteria: No more than 10 percent of any group is lost due to
autolysis, cannibalism, or management problems; and survival in each
group is no less than 50 percent at 15 months for mice and 18 months
for rats. Survival should not fall below 25 percent at 18 months for mice
and 24 months for rats.
(iv) The use of historical control data from an appropriate time period
from the same testing laboratory (i.e., the incidence of tumors and other
suspect lesions normally occurring under the same laboratory conditions
and in the same strain of animals employed in the test) is helpful for as-
sessing the significance of changes observed in the current study.
(3) Test report, (i) In addition to the reporting requirements as speci-
fied under 40 CFR part 792, subpart J, 40 CFR part 160, and the OECD
Principles of GLP (ISBN 92-64-12367-9), the following specific informa-
tion should be reported:
(A) Test substance characterization should include:
(7) Chemical identification.
(2) Lot or batch number.
(3) Physical properties.
(4) Purity/impurities.
(5) Identification and composition of any vehicle used.
(B) Test system should contain data on:
(7) Species and strain of animals used and rationale for selection if
other than that recommended.
(2) Age including body weight data and sex.
(3) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods.
(4) Identification of animal diet.
(5) Acclimation period.
(C) Test procedure should include the following data:
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(7) Method of randomization used.
(2) Full description of experimental design and procedure.
(3) Dose regimen including levels, methods, and volume.
(4) Test results, (i) Group animal data. Tabulation of toxic response
data by species, strain, sex and exposure level for:
(A) Number of animals exposed.
(B) Number of animals showing signs of toxicity.
(C) Number of animals dying.
(ii) Individual animal data. Data should be presented as summary
(group mean) as well as for individual animals.
(A) Time of death during the study or whether animals survived to
termination.
(B) Time of observation of each abnormal sign and its subsequent
course.
(C) Body weight data.
(D) Feed and water consumption data, when collected.
(E) Achieved dose (mg/kg/day) as a time-weighted average if the test
substance is administered in the diet or drinking water.
(F) Results of clinical pathology when performed.
(G) Necropsy findings including absolute/relative organ weight data.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(J) Historical control data.
(iii) In addition, for inhalation studies the following should be re-
ported:
(A) Test conditions. The following exposure conditions must be re-
ported.
(7) Description of exposure apparatus including design, type, dimen-
sions, source of air, system for generating particulate and aerosols, method
of conditioning air, treatment of exhaust air and the method of housing
the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and particu-
late aerosol concentrations and size should be described.
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(B) Exposure data. These should be tabulated and presented with
mean values and a measure of variability (e.g. standard deviation) and
should include:
(7) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Actual (analytical or gravimetric) concentration in the breathing
zone.
(4) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(5) Particle size distribution, calculated MMAD and geometric stand-
ard deviation (GSD).
(6) Explanation as to why the desired chamber concentration and/
or particle size could not be achieved (if applicable) and the efforts taken
to comply with this aspect of the guidelines.
(f) Quality assurance. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment. The study must be conducted in compliance with the GLP reg-
ulations as described by the Agency (40 CFR parts 160 and 792) and
the OECD Principles of GLP (ISBN 92-64-12367-9).
(g) References. The following references should be consulted for ad-
ditional background information on this guideline:
(1) Benitz, K.F. Measurement of Chronic Toxicity. Methods of Toxi-
cology. Ed. G.E. Paget. Blackwell, Oxford, pp. 82-131 (1970).
(2) D'Aguanno, W. Drug Safety Evaluation—Pre-Clinical Consider-
ations. Industrial Pharmacology: Neuroleptic. Vol. I. Ed. S. Fielding and
H. Lai. Futura, Mt. Kisco, NY. pp. 317-332 (1974).
(3) Department of Health and Welfare. The Testing of Chemicals for
Carcinogenicity, Mutagenicity, Teratogenicity. Minister of Health and
Welfare. Department of Health and Welfare, Canada (1975).
(4) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety
of Chemicals in Foods, Drugs and Cosmetics. The Association of Food
and Drug Officials of the United States, pp. 36-45 (1959, 3rd Printing
1975).
(5) Food Safety Council. Proposed System for Food Safety Assess-
ment. Prepared by the Scientific Committee, Food Safety Council. Food
and Cosmetic Toxicology , Vol. 16, Supplement 2 (December 1978).
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(6) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, Ap-
praisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The
Association of Food and Drug Officials of the United States, pp. 60-67
(1959, 3rd Printing 1975).
(7) International Union Against Cancer. Carcinogenicity Testing.
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