United States Prevention, Pesticides EPA712-C-98-212
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.4300
Combined Chronic
Toxicity/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 870.4300 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-
ies.
(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-
line.
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-
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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.
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(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-
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-
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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.
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(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).
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(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
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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
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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-
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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-
corded.
(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.
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(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-
ommended.
(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
cholinesterases.
(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.
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(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
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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 these data may pro-
vide significant guidance to the pathologist.
(12) Histopathology. (i) The following histopathology should be per-
formed:
(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
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changes, effects on mortality and any other general or specific toxic ef-
fects.
(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 (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-
ported:
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(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 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
zone.
(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)
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(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).
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(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
(1976)
(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,
D.C.
(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.
(1977).
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(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).
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