United States       Prevention, Pesticides     EPA712-C-98-212
          Environmental Protection    and Toxic Substances     August 1998
          Agency         (7101)
&EPA    Health Effects Test
          OPPTS 870.4300
          Combined Chronic

     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 870.4300  Combined chronic toxicity/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 798.3320 Combined Chronic
Toxicity/Oncogenicity;   OPP   83-5   Combined   Chronic    Toxicity/
Oncogenicity (Pesticide Assessment Guidelines, Subdivision  F—Hazard
Evaluation; Human and Domestic Animals) EPA report 540/09-82-025,
1982; and OECD 453 Combined Chronic Toxicity/Carcinogenicity Stud-

     (b) Purpose. The objective of a combined chronic toxicity/carcino-
genicity study is to determine the effects of a substance in a mammalian
species following prolonged and repeated exposure. The application of this
guideline should generate data which identify the majority of chronic and
carcinogenicity effects and  determine dose-response relationships. The de-
sign and conduct should allow for the detection of neoplastic  effects and
a determination of the carcinogenic potential as well  as general toxicity,
including neurological, physiological, biochemical,  and  hematological ef-
fects and exposure-related morphological (pathology) effects.

     (c) Definitions. The definitions in section 3 of TSCA and the  defini-
tions in 40 CFR Part 792—Good Laboratory  Practice Standards  (GLP)
apply to this guideline. The following definitions also  apply to this guide-

     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.

     Chronic  toxicity  is the adverse effects occurring 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 combined  chronic toxicity/carcinogenicity  study  is the
amount of test  substance administered via the oral, dermal,  or inhalation
routes for a period of up to 24  months.  Dose  is expressed  as weight of
the test substance per  unit body weight of test animal (milligrams per kilo-
gram),  or as  weight of the test  substance in parts  per million  (ppm) in
food or drinking 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 per day. For dermal application, dose is ex-

pressed as weight of the test substance (grams, milligrams) per unit body
weight of the test animal (milligrams per kilogram) or as weight of the
substance per unit surface area (milligrams per square centimeter) per day.

    No-observed-effect-level (NOEL) is the  maximum  dose used  in  a
study which produces no observed adverse effects.  The NOEL is usually
expressed in  terms of the weight of a test substance given daily per unit
weight of test animal (milligrams per kilogram per day).

    Target organ is any organ of a test animal  showing evidence of an
effect induced by a test substance.

    (d) Limit test.  If a  test at one dose level of at  least 1,000 mg/kg
body weight (expected human exposure may indicate the need for a higher
dose level),  using the procedures described for  this study, produces no
observable toxic effects  or if toxic effects would not  be  expected based
upon data of structurally related compounds, then a full study using three
dose levels might not be necessary.

    (e) Test procedures—(1) Animal selection—(i) Species and  strain.
Preliminary studies providing data on acute,  subchronic, and metabolic re-
sponses should have been carried out to permit an appropriate choice of
animals (species and strain).  As discussed in other  guidelines, the mouse
and rat have been most widely used for assessment of carcinogenic poten-
tial, while the rat and dog have been most often studied for chronic tox-
icity. For the combined chronic toxicity/carcinogenicity study via the oral
and inhalation routes, the rat is the species  of choice and for the  dermal
route, the mouse is species of choice. If other species are used, the tester
should provide justification/reasoning for their  selection. The strain se-
lected should be susceptible to the carcinogenic or toxic effect of the class
of substances being tested, if known,  and provided it does not have a spon-
taneous background incidence too high for meaningful assessment. Com-
monly used laboratory strains should be employed.

    (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. At least
20 additional rodents (10 males and 10 females) should be used for sat-
ellite dose groups and the satellite control group. The purpose of the sat-
ellite group is  to allow  for  the evaluation of chronic toxicity after 12
months of exposure to the test substance.

     (B)  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.

     (C)  To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required.

     (D)  Each animal should be assigned  a unique identification number.
Dead animals  (and 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.
Rodents  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 nor substantially alter
the chemical or toxicological  properties of the test substance.  It is rec-
ommended that wherever possible the usage of an aqueous solution be
considered first, followed by consideration of a solution in oil, and finally
solution in other vehicles.

     (ii) One lot of the test substance should be used throughout the  dura-
tion of the  study  if possible,  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 is required. 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.  If the
toxic properties of the  vehicle are not known  or cannot be made available,
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 in rodents should elicit signs  of toxicity
without substantially 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 assess the chron-
ic toxicity and the  carcinogenic potential of the test substance.  Thus,  the
selection of the highest dose to be tested is dependent upon changes  ob-
served in several toxicological  parameters in subchronic  studies. The  high-
est 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).
     (9) Inflammation (moderate).
     (C) Gross criteria for exceeding the high dose:
     (7) Ulcers-fissures, exudate/crust (eschar),nonviable (dead) tissues.
     (2) 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.
        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) Preparation  of animal  skin.  Shortly before
testing, fur  should be clipped from not less than 10  percent of the  body
surface area for application of the test substance. In order to dose approxi-
mately  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.  Shaving should be
carried out  approximately 24  hours before dosing. Repeated clipping or
shaving is usually needed at approximately weekly intervals. When clip-
ping or shaving the fur, care should be taken to avoid abrading the skin
which could alter its permeability.

     (B) 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.

     (C) Administration of test substance. The duration of exposure should
be at least 18 months for mice and hamsters and 24 months for rats. Ideal-
ly,  the animals should be treated with test substance for at least 6 h/day
on  a 7-day per week basis. However, based on practical considerations,
application  on  a 5-day per week  basis  is acceptable. Dosing should be
conducted at approximately the same time each day. The test substance
should be applied uniformly over the treatment site.The surface  area cov-
ered may be less for highly toxic substances. As much of the area should

be covered with as thin and uniform a film as possible. For rats, the test
substance may be held in contact with the skin with a porous gauze dress-
ing and nonirritating tape if necessary. The test site should be further cov-
ered in a suitable manner to retain the gauze dressing plus test substance
and to ensure that the animals cannot ingest the test substance. The appli-
cation site should not be covered when the mouse is the species of choice.
The test substance may be wiped from the skin after the 6-hour exposure
period 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 pri-
marily 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. It is  not normally necessary to measure chamber oxygen con-
centration if airflow is adequate.

     (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, 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. The animals should be acclimated
and heat stress minimized.

     (D) The temperature at which the test is performed  should be main-
tained at 22 + 2 °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 the 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 shall be measured
in the animal's breathing  zone. During the exposure period, the actual con-
centrations of the test substance shall  be held as constant as practicable
and monitored continuously or intermittently depending on the method of

analysis. Chamber concentration may be measured using gravimetric or
analytical methods as appropriate. If trial run measurements are reasonably
consistent (±10 percent for liquid aerosol, gas, or vapor; + 20 percent
for dry  aerosol), then two measurements should be sufficient.  If measure-
ments are not  consistent, 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 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 in measuring
chamber analytical concentration due to precipitation, nonhomogeneous
mixtures, volatile components, or other factors, additional analyses 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. The mass median aerodynamic 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.  Meas-
urements of aerodynamic  particle size  in the  animal'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 sufficient. If pretest measurements are not  within
10  percent of each  other,  three  to four  measurements should be  taken.

     (I)  Feed should be withheld during exposure. Water may also be with-
held during exposure.

     (6) Observation period,  (i) 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 duration of the study is advised.

     (ii) Animals in  a satellite group to  assess chronic toxicity should be
observed for 12 months.

     (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 to the study (e.g., necropsy or  refrig-
eration  of those animals found dead and isolation or sacrifice of weak
or moribund animals). General  clinical  observations should  be made at
least once a  day, preferably at the same time each  day, taking  into consid-


eration  the peak period of anticipated effects  after dosing.  The  clinical
condition of the animal should be recorded.

     (ii) A careful clinical examination should be made at least once prior
to the initiation of treatment (to allow for within subject comparisons) and
once weekly during treatment in all animals. These observations should
be made outside the home cage, preferably in a  standard arena, and  at
similar  times on each occasion. Effort should be made to ensure that vari-
ations in the observation conditions are minimal. Observations should be
detailed and carefully recorded, preferably using scoring systems, explic-
itly defined by the testing laboratory.  Signs  noted  should include,  but not
be limited  to, changes in skin, fur, eyes, mucous membranes, occurrence
of secretions  and excretions  and autonomic activity (e.g.,  lacrimation,
piloerection, pupil size, unusual respiratory pattern). Changes in gait, pos-
ture  and response to handling as well as the presence of clonic or tonic
movements, stereotypies (e.g., excessive grooming, repetitive circling)  or
bizarre  behavior (e.g., self-mutilation  , walking backwards) should be re-

     (iii) Once, near the end of the first year of the exposure period and
in any  case not earlier than in month 11, assessment of motor activity,
grip  strength, and sensory reactivity to stimuli of different types (e.g., vis-
ual, auditory, and proprioceptive stimuli) should be conducted in rodents.
Further  details  of the procedures  that could  be followed are  described  in
the references  listed  under  paragraphs (h)(2), (h)(7),  (h)(9), (h)(12),
(h)(13), and (h)(25) of this guideline.

     (iv) Functional  observations  conducted  towards the end  of the study
may be omitted when data  on functional observations are available from
other studies and the daily clinical observations did not reveal any func-
tional deficits.

     (v) Exceptionally, functional observations may be omitted for groups
that otherwise reveal signs of toxicity to an extent that would significantly
interfere with functional test performance.

     (vi) Body  weights  should be recorded individually  for all animals
once prior  to administration of the test substance, 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 facili-
tate monitoring of health status.

     (vii) Measurements of feed consumption should be determined weekly
during the  first 13 weeks of the study and then at  approximately monthly
intervals unless health status or body weight changes dictate otherwise.
Measurements  of water consumption should be determined  at the  same
intervals if the test material is administered in drinking water.

     (viii) Moribund animals should be removed and sacrificed when no-
ticed and the time of death should be recorded  as precisely as possible.
At the  end of the study period, all survivors should be sacrificed. Animals
in the  satellite  group should  be  sacrificed after 12 months of exposure
to the test substance (interim sacrifice).

     (8)   Clinical  pathology.  Hematology,   clinical   chemistry  and
urinalyses should be performed from 10 animals per sex per group.  The
parameters should be  examined at approximately  6 month intervals during
the first  12 months of the study. If possible, these collections should be
from the same animals at each interval. If hematological and biochemical
effects are seen in the subchronic study, testing should also be performed
at 3  months. Overnight fasting of animals  prior to blood sampling is  rec-

     (i) Hematology.  The recommended  parameters are red blood  cell
count,  hemoglobin concentration, hematocrit, mean corpuscular  volume,
mean corpuscular hemoglobin, and mean corpuscular hemoglobin con-
centration, white blood cell  count,  differential  leukocyte count, platelet
count,  and a measure of  clotting potential, such as prothrombin time or
activated partial thromboplastin time.

     (ii) Clinical  chemistry. (A) Parameters which are considered appro-
priate to all studies are electrolyte balance, carbohydrate metabolism,  and
liver and kidney function.  The selection of specific tests will be influenced
by observations on the mode of action of the substance and signs of clini-
cal toxicity.

     (B)  The recommended clinical chemistry determinations are  potas-
sium, sodium, glucose, total  cholesterol,  urea nitrogen,  creatinine, total
protein, and albumin. More than two hepatic enzymes,  (such as alanine
aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol
dehydrogenase, or gamma glutamyl  transpeptidase) should also be  meas-
ured. Measurements of addtional  enzymes (of hepatic or other origin) and
bile acids, may also be useful.

     (iii) If a test chemical has  an  effect on the hematopoietic system,
reticulocyte counts and bone marrow cytology may be indicated.

     (iv) Other determinations that should be carried out if the test chemi-
cal is known or suspected of affecting related measures include calcium,
phosphorus,  fasting   triglycerides,   hormones,  methemoglobin,   and

     (v) Urinalyses. Urinalysis for rodents should be performed at the end
of the first year of the study using timed urine collection. Urinalysis deter-
minations include: appearance, volume, osmolality or specific gravity,  pH,
protein, glucose, and blood/blood  cells.


     (9) Ophthalmological examination.  Examinations should be  made
on all animals using an ophthalmoscope or an equivalent  device prior to
the administration of the test substance and  at termination of the  study
on  10  animals per sex in the high-dose and control groups.  If changes
in eyes are detected, all animals should be examined.

     (10)  Gross necropsy, (i) A  complete gross examination should  be
performed on all animals, including those which died during the experi-
ment or were killed in a moribund condition.

     (ii) At least, the liver, kidneys, adrenals, testes, epididymides, ovaries,
uterus, spleen, brain, and heart should be trimmed and 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 these data may pro-
vide significant guidance to the pathologist.

     (12) Histopathology. (i) The following  histopathology should be per-

     (A) Full histopathology on the organs and tissues,  listed  under  para-
graph (e)(10)(iii) of this guideline of all animals in the control and high
dose groups and of 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  levels  should be  examined fully  as described in paragraph
(e)(12)(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.

     (f) 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 combined
chronic toxicity/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 will
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 ef-

     (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 (milligrams/kilogram body weight) as  a  time-
weighed average is  the test substance is administered in the diet or drink-
ing water.

     (F) Results of ophthalmological examination, when performed.

     (G) Results of hematological tests performed.

     (H) Results of clinical chemistry tests performed.

     (I) Results of urinalysis tests performed.

     (J) Results of observations made.

     (K) Necropsy findings including absolute/relative organ weight data.

     (L) Detailed description of all histopathological findings.

     (M) Statistical treatment of results where appropriate.

     (N) 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 particulates and aerosols, meth-
od 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, and calculated mass median aerodynamic
diameter  (MMAD) and geometric standard 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.

     (g) 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).

     (h) 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) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W.,
Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity
Experiments:   Implication    for    Neurotoxicological    Assesments.
Neurotoxicol. Teratol. 13, 599-609. (1991)


     (3) 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).

     (4) 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).

     (5) 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).

     (6) Food Safety Council. Proposed system for food safety assessment.
Prepared by the scientific committee, Food Safety Council. Food and Cos-
metic Toxicology, Vol. 16, Supplement 2. (December 1978).

     (7) Gad S.C. A Neuromuscular  Screen for Use in Industrial Toxi-
cology. J Toxicol Environ. Health, 9, 691-704. (1982)

     (8) 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).

     (9)  International Programme on Chemical  Safety. Principles and
Methods for the  Assessment of Neurotoxicity Associated with Exposure
to Chemicals. Environmental Health Criteria Document No. 60. (1986)

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

     (11) 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, Ohio (September 1975).

     (12) Meyer  O.A., Tilson H.A., Byrd W.C.,  Riley  M.T.  A Method
for the Routine Assessment of Fore- and Hind-Limb Grip Strength of Rats
and Mice. Neurobehav. Toxicol. 1, 233-236. (1979)

     (13) Moser V.C., McDaniel K.M., Phillips P.M. Rat Strain and Stock
Comparisons using a Functional Observational  Battery:  Baseline Values
and  Effects  of Amitraz. Toxicol.  Appl. Pharmacol.  108, 267-283 (1991)

     (14) 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).


     (15) 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

     (16) 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).

     (17)  Organization  for  Economic  Cooperation  and  Development.
Guidelines for Testing of Chemicals,  Section 4-Health  Effects, Part 453
Combined Chronic Toxicity/Carcinogenicity Studies, Paris. (1981).

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

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

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

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

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

     (23) The Atlas  Of Dermal Lesions, EPA Report 20T-004,  U.S  Envi-
ronmental Protection Agency, Washington, D.C.

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

     (25) Tupper, D.E., Wallace  R.B. Utility of the Neurologic Examina-
tion in Rats. Acta. Neurobiol. Exp. 40, 999-1003 (1980).

     (26) United States Environmental Protection Agency. Office of Test-
ing and Evaluation.  Proposed Halth Effects Test Standards for Toxic Sub-
stances Control Act Test Rules. 40 CFR Part 772. Standard for Develop-
ment of Test Data. Subpart D. Chronic  Health Effects. FEDERAL REGISTER.
Vol.  44, No.91. pp. 27350-27362.

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


     (28) Weingand, K., Brown, G., Hall, R. et al. Harmonization of Ani-
mal  Clinical Pathology Testing in Toxicity and Safety Studies. Fundam.
&Appl. Toxicol. 29:198-201. (1996)

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

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

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