United States       Prevention, Pesticides     EPA 712-C-98-211
           Environmental Protection    and Toxic Substances     August 1998
           Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.4200

     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

     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

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

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.

     (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-

     (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

     (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.

     (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).

     (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.

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

     (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

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.

     (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


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-

     (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

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-

     (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),


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:


     (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
     (B) Time of observation of each abnormal sign and its  subsequent
     (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-
     (A) Test conditions. The following exposure conditions must be re-
     (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.

     (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

     (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

     (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).


     (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.
UCC Technical  Report Series,  Vol.2.  Ed.  I.  Berenblum. International
Union Against Cancer, Geneva (1969).

     (8) Leon, B.K.J. and Laskin, S. Number and Species of Experimental
Animals for Inhalation Carcinogenicity Studies. Paper presented at Con-
ference  on Target Organ Toxicity. Cincinnati, OH (September 1975).

     (9) National Academy of Sciences. Principles and Procedures for
Evaluating the Toxicity of Household  Substances. A report prepared by
the Committee for the Revision of NAS Publication 1138, under the aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences. Washington, DC (1977).

     (10) National  Cancer Institute. Report of the Subtask Group on Car-
cinogen Testing to the Interagency Collaborative Group on Environmental
Carcinogenesis. United  States National Cancer Institute. Bethesda, MD

     (11) National Center for Toxicological Research. Appendix B, Report
of Chronic Studies  Task Force Committee, April 13-21, 1972. National
Center for Toxicological Research, Rockville, MD (1972).

     (12)  Organization  for Economic  Cooperation  and  Development.
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 451
Carcinogenicity Studies, Paris (1981).

     (13)  Page, N.P.  Chronic Toxicity and Carcinogenicity Guidelines.
Journal of Environmental  Pathology  and Toxicology.  11:161-182 (1977).

     (14) Page, N.P. Concepts of a Bioassay Program in Environmental
Carcinogenesis, Advances  in Modern Toxicology. Vol.3, Ed. Kraybill and
Mehlman. Hemisphere, Washington, DC. pp. 87-171 (1977).

     (15) Schwartz, E. Toxicology of Neuroleptic Agents. Industrial Phar-
macology: Neuroleptics. S. Fielding  and H. Lai. Futura, Mt. Kisco, NY.
pp. 203-221 (1974).

     (16) Sontag, J.M. et al. Guidelines for Carcinogen Bioassay in Small
Rodents. NCI-CS-TR-1 United States Cancer Institute, Division of Cancer
Control  and Prevention,  Carcinogenesis Bioassay Program. Bethesda,  MD.

     (17) Summary of the  EPA Workshop on Carcinogenesis Bioassay via
the Dermal Route. EPA Report 50/6-89-002; 50/6-89-003. Washington,


    (18) The Atlas of Dermal Lesions, EPA Report 20t-2004, U.S Envi-
ronmental Protection Agency, Washington, DC.

    (19) Toxicity and Clinical Trial Subcommittee, Committee on Safety
of Medicine, November, 1977.

    (20) United States Environmental Protection Agency. Office of Test-
ing  and Evaluation. Proposed health  effects test  standards for toxic  sub-
stances control act test rules. 40 CFR Part 772. Standard for Development
of Test Data. Subpart  D. Chronic Health Effects. FEDERAL REGISTER. No.
91 (44 FR 27350-27362).

    (21) United States Pharmaceutical Manufacturers Association. Guide-
lines for the Assessment of Drug and Medical Device Safety in Animals.

    (22) World Health Organization (WHO). Guidelines for Evaluation
of Drugs for Use in Man. WHO Technical Report Series No. 563. (WHO),
Geneva (1975).

    (23) World Health Organization (WHO). Part I. Environmental Health
Criteria 6. Principles and Methods for Evaluating the Toxicity of Chemi-
cals. (WHO), Geneva (1978).

    (24) World Health Organization (WHO). Principles for  Pre-Clinical
Testing of Drug Safety. WHO Technical Report Series No. 341. (WHO),
Geneva (1966).