United States Prevention, Pesticides EPA712-C-98-210
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.4100
Chronic Toxicity
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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, chap-
ter I, subchapter R of the Code of Federal Regulations (CFR), the Office
of Pesticide Programs (OPP) which appeared in publications of the Na-
tional Technical Information Service (NTIS) and the guidelines published
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. I36,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 870.4100 Chronic toxicity.
(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 798.3260 Chronic Toxicity;
OPP 83-1 Chronic Feeding—Two Species, Rodent and Nonrodent (Pes-
ticide Assessment Guidelines, Subdivision F—Hazard Evaluation; Human
and Domestic Animals) EPA report 540/09-82-025, 1982; and OECD 452
Chronic Toxicity studies.
(b) Purpose. The objective of a chronic toxicity study is to determine
the effects of a substance in a mammalian species following prolonged
and repeated exposure. A chronic toxicity study should generate data from
which to identify the majority of chronic effects and to define long-term
dose-response relationships. The design and conduct of chronic toxicity
tests should allow for the detection of general toxic effects, including neu-
rological, physiological, biochemical, and hematological effects and expo-
sure-related morphological (pathological) effects.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definitions also apply to this test guideline.
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 doses occurring
as a result of prolonged action on, or increased concentration of, the ad-
ministered test substance or its metabolites in susceptible tissue.
Dose in a chronic toxicity study is the amount of test substance ad-
ministered daily via the oral, dermal or inhalation routes for a period of
at least 12 months. Dose is expressed as weight of the test substance
(grams, milligrams) per unit body weight (BW) of test animal (milligram
per kilogram), or as weight of the test substance in parts per million (ppm)
in food or drinking water per day. For inhalation exposure, dose is ex-
pressed as weight of the test substance per unit volume of air (milligrams
per liter) or as parts per million per day. For dermal exposure, dose is
expressed 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 of surface area (milligrams per square centimeter) per
day.
No-observed-effect-level (NOEL) is the maximum dose used in a
study which produces no adverse effects. The NOEL is usually expressed
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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
BW (expected human exposure may indicate the need for a higher dose
level), using the procedures described for this study, produces no observ-
able toxic effects and if toxicity would not be expected based upon data
of structurally related compounds, a full study using three dose levels
might not be necessary.
(e) Test procedures—(1) Animal selection—(i) Species and strain.
Testing should be performed with two mammalian species, one a rodent
and the other a nonrodent. The rat is the preferred rodent species and the
dog is the preferred nonrodent species. Commonly used laboratory strains
should be employed. 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 of rodents should generally begin no later than 8 weeks
of age.
(C) Dosing of dogs should begin between 4 and 6 months of age
and in no case later than 9 months of age.
(D) At commencement of the study, the weight variation of animals
used should be within 20 percent of the mean weight for each sex.
(E) 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) For rodents, at least 40 animals (20 males and
20 females) and for nonrodents (dogs) at least 8 animals (4 females and
4 males) 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) The number of animals at the termination of the study must be
adequate for a meaningful and valid statistical evaluation of chronic ef-
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fects. The Agency must be notified if excessive early deaths or other prob-
lems are encountered that might compromise the integrity of the study.
(D) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required.
(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) Rodents 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. Caging should be appropriate to the nonrodent
species. However, it is recommended that dogs are housed individually.
(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 adequacy of nutritional require-
ments 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 5 days is rec-
ommended.
(2) Control and test substances, (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle. If a vehicle or dilu-
ent is needed it should not elicit toxic effects itself nor substantially alter
the chemical or toxicological properties of the test substance. It is rec-
ommended that wherever possible the use of an aqueous solution be the
first choice, followed by consideration of solution in oil, and finally, solu-
tion in other vehicles.
(ii) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability. Prior to the initiation
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of the study, there should be a characterization of the test substance, in-
cluding the purity of the test compound, and, if technically feasible, the
names 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) Satellite group. A satellite group of 40 animals (20 animals per
sex) for rodents and 8 animals (4 animals per sex) for nonrodents may
be treated with the high-dose level for 12 months and observed for revers-
ibility, persistence, or delayed occurrence of toxic effects for a post-treat-
ment of appropriate length, normally not less than 28 days. In addition,
a control group of 40 animals (20 animals per sex) for rodents and 8 ani-
mals (4 animals per sex) for nonrodents should be added to the satellite
study.
(5) Dose levels and dose selections, (i) In chronic toxicity tests, it
is desirable to determine a dose-response relationship as well as a NOEL.
Therefore, at least three dose levels with a control group and, where appro-
priate, a vehicle control (corresponding to the concentration of the vehicle
at the highest exposure level) should be used. Dose levels should be spaced
to produce a gradation of effects. A rationale must be provided for the
doses selected.
(ii) The highest-dose level should elicit signs of toxicity without sub-
stantially altering the normal life span of the animal. 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 chronic toxicity of the test
substance. Thus, the selection of the highest dose to be tested is dependent
upon changes observed in several toxicological parameters in subchronic
studies. The highest dose tested need not exceed 1,000 mg/kg/day. If der-
mal application of the test substance produces severe skin irritation, then
it may be necessary either to terminate the study and choose a lower high-
dose level or to reduce the dose level. Gross criteria for defining severe
irritation would include ulcers, fissures, exudate/crust(eschar), dead tissue,
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or anything leading to destruction of the functional integrity of the epider-
mis (e.g. caking, open sores, fissuring, eschar). Histological criteria for
defining severe irritation would include follicular and interfollicular crust,
microulcer, mild/moderate degeneration/necrosis, moderate/marked epi-
dermal edema, marked dermal edema, and marked inflammation.
(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.
(6) 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. Ideally, the animals should be dosed by gavage or
with capsules on a 7-day per week basis for a period of at least 12 months.
However, based primarily on practical considerations, dosing by gavage
or capsules on a 5-day per week schedule is acceptable. If the test sub-
stance is administered via in the drinking water or mixed in the diet, expo-
sure 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)(5)(ii) 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 (j,L 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 for 12 months. Ideally, 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 ac-
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ceptable. 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 covered 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 dressing and nonirritating tape if necessary. The
test site should be further covered in a suitable manner to retain the gauze
dressing plus test substance and to ensure that the animals cannot ingest
the test substance. The application site should not be covered when the
mouse is the species of choice. The test substance may be wiped from
the skin after the six-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 12 months. However, based primarily on practical considerations, ex-
posure for 6 hours per 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. When 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 Sit 22 ±2 °C. The relative humidity should be maintained between
40-60 percent, but in certain instances (e.g., 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 min.
(G) The actual concentrations of the test substance should be meas-
ured in the breathing zone. During the exposure period, the actual con-
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centrations of the test substance should be held as constant as practicable,
monitored continuously or intermittently depending on the method of anal-
ysis. Chamber concentration may be measured using gravimetric or analyt-
ical methods, as appropriate. If trial run measurements are reasonably con-
sistent (±10 percent for liquid aerosol, gas, or vapor; + 20 percent for
dry aerosol), then two measurements should be sufficient. If measurements
are not consistent, three to four measurements should be taken. Whenever
the test substance is a formulation, or it is necessary to formulate the test
substance with a vehicle for aerosol generation, the analytical concentra-
tion must be reported for the total formulation, and not just for the active
ingredient (AI). If, for example, a formulation contains 10 percent 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 pro-
vided in the study report. If there is some difficulty measuring chamber
analytical concentration due to precipitation, nonhomogeneous mixtures,
volatile components, or other factors, additional analysis of inert compo-
nents 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.
(7) Observation period, (i) Animals should be observed for a period
of at least 12 months.
(ii) Animals in a satellite group (if used) scheduled for follow-up ob-
servations should be kept for at least 28 days further without treatment
to detect recovery from, or persistence of, toxic effects.
(8) 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)(6), (h)(8), (h)(9), (h)(10),
and (h)(17) 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 the administration of the test substance, once a week during
the first 13 weeks of 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 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.
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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.
All survivors should be sacrificed at the end of the study period.
(9) Clinical pathology. Hematology, clinical chemistry, and urinal-
ysis should be performed on 10 rats per sex per group, and on all non-
rodents. In rodents, the parameters should be examined at approximately
6 month intervals during the conduct of the study and at termination. If
possible, these collections should be from the same animals at each inter-
val. In nonrodents, the parameters should be examined once or twice prior
to initiation of treatment, at 6-month intervals during the conduct of the
study, and at termination. If hematological and biochemical effects were
seen in the subchronic study, testing should also be performed at 3 months.
Overnight fasting of animals prior to blood sampling is recommended.
(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, calcium (nonrodent), phosphorus (nonrodent), chloride
(nonrodent), glucose, total cholesterol, urea nitrogen, creatinine, total pro-
tein, total bilirubin (nonrodent), and albumin. More than two hepatic en-
zymes, (such as alanine aminotransferase, aspartate aminotransferase, alka-
line phosphatase, sorbitol dehydrogenase, or gamma glutamyl
transpeptidase) should also be measured. Measurements of addtional en-
zymes (of hepatic or other origin) and bile acids, may also be useful.
(C) If a test chemical has an effect on the hematopoietic system,
reticulocyte counts and bone marrow cytology may be indicated.
(D) 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.
(iii) Urinalysis. Urinalysis for rodents should be performed at the
end of the study using timed urine collection. Urinalysis for nonrodents
should be performed prior to treatment, midway through treatment and
at the end of the study using timed urine collection. Urinalysis determina-
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tions include: appearance, volume, osmolality or specific gravity, pH, pro-
tein, glucose, and blood/blood cells.
(10) Ophthalmological examination. Examinations should be made
of all animals using an ophthalmoscope or equivalent device prior to the
administration of the test substance and at termination of the study on
10 rats of each sex in the high-dose and control groups and preferably
in all nonrodents, but at least the control and high-dose groups should
be examined. If changes in eyes are detected, all animals should be exam-
ined.
(11) Gross necropsy, (i) All animals should be subjected to a full
gross necropsy which includes examination of the external surface of the
body, all orifices, and the cranial, thoracic and abdominal cavities and their
contents.
(ii) At least the liver, kidneys, adrenals, testes, epididymides, ovaries,
uterus, nonrodent thyroid (with parathyroid), 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.
(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.
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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 considered 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.
(12) Histopathology. (i) The following histopathology should be per-
formed:
(A) Full histopathology on the organs and tissues (listed under para-
graph (e)(ll)(iii) of this guideline) of all rodents and nonrodents in the
control and high-dose groups, and all rodents and nonrodents that died
or were killed during the study. The examination should be extended to
all animals in all dosage groups if treatment-related changes are observed
in the high-dose group.
(B) All gross lesions in all animals.
(C) Target tissues in all animals.
(ii) If the results show substantial alteration of the animal's normal
life span, or other effects that might compromise the significance of the
data, the next lower levels should be examined fully as described in para-
graph (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.
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(2) Evaluation of study results. The findings of a chronic toxicity
study should be evaluated in conjunction with the findings of preceding
studies and considered in terms of the toxic effects as well as 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 effects.
(3) Test report. In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J, 40 CFR part 160, and the OECD Prin-
ciples of GLP (ISBN 92-64-12367-9), the following specific information
should be reported:
(i) Test substance characterization should include:
(A) Chemical identification.
(B) Lot or batch number.
(C) Physical properties.
(D) Purity/impurities.
(ii) Identification and composition of any vehicle used.
(iii) Test system should contain data on:
(A) Species and strain of animals used and rationale for selection
if other than that recommended.
(B) Age including body weight data and sex.
(C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods.
(D) Identification of animal diet.
(E) Acclimation period.
(iv) Test procedure should include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regimen including levels, methods, and volume.
(v) Test results.
(A) Group animal data. Tabulation of toxic response data by species,
strain, sex and exposure level for:
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(7) Number of animals exposed.
(2) Number of animals showing signs of toxicity.
(3) Number of animals dying.
(B) Individual animal data. Data should be presented as summary
(group mean) as well as for individual animals.
(7) Time of death during the study or whether animals survived to
termination.
(2) Time of observation of each abnormal sign and its subsequent
course.
Body weight data.
(4) Feed and water (if collected) consumption data.
(5) Achieved dose (mg/kg/day) as a time-weighted average if the test
substance is administered in the diet or drinking water.
(6) Results of ophthalmological examinations.
(7) Results of hematological tests performed.
(8) Results of clinical chemistry tests performed.
(9) Urinalysis tests performed and results.
(Iff) Results of observations made.
(11} Necropsy findings, including absolute and relative (to body
weight) organ weight data.
(72) Detailed description of all histopathological findings.
(13) Statistical treatment of results, where appropriate.
(iv) In addition, for inhalation studies the following should be re-
ported:
(A) Test conditions. The following exposure conditions must be re-
ported:
(7) Description of exposure apparatus including design, type, dimen-
sions, source of air, system for generating particulate and aerosols, method
of conditioning air, treatment of exhaust air and the method of housing
the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and particu-
late aerosol concentrations and size should be described.
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(B) Exposure data. These data should be tabulated and presented with
mean values and a measure of variability (e.g., standard deviation) and
should include:
(7) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Actual (analytical or gravimetric) concentration in the breathing
zone.
(4) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(5) Particle size distribution, calculated MMAD, and geometric stand-
ard deviation (GSD).
(6) Explanation as to why the desired chamber concentration and/
or particle size could not be achieved (if applicable) and the efforts taken
to comply with this aspect of the guidelines.
(g) Quality control. 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 GLP regula-
tions 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 test 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) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety
of Chemicals in Foods, Drugs and Cosmetics. The Association of Food
and Drug Officials of the United States, pp. 36-45 (1959, 3rd Printing
1975).
(5) Food Safety Council. Proposed System for Food Safety Assess-
ment. Prepared by the Scientific Committee, Food Safety Council. Food
and Cosmetic Toxicology. Vol. 16, Supplement 2. (December 1978).
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(6) Gad S.C. A Neuromuscular Screen for Use in Industrial Toxi-
cology. Journal of Toxicology and Environmental Health. 9, 691-704.
(1982)
(7) 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).
(8) 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)
(9) 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)
(10) 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)
(11) 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 auspices
of the Committee on Toxicology, National Research Council, National
Academy of Sciences, Washington, DC (1977).
(12) 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).
(13) Organization for Economic Cooperation and Development.
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 452
Chronic Toxicity Studies, Paris (1981).
(14) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines.
Journal of Environmental Pathology and Toxicology. 11:161-182 (1977).
(15) Schwartz, E. Toxicology of Neuroleptic Agents. Industrial Phar-
macology: Neuroleptic. S. Fielding and H. Lai. Futura, Mt. Kisco, NY
pp. 203-221 (1974).
(16) Toxicity and Clinical Trial Subcommittee, Committee on Safety
of Medicine. (November 1977).
(17) Tupper, D.E., Wallace R.B. Utility of the Neurologic Examina-
tion in Rats. Acta. Neurobiol. Exp. 40, 999-1003 (1980).
(18) United States Pharmaceutical Manufacturers Association. Guide-
lines for the Assessment of Drug and Medical Device Safety in Animals.
(1977).
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(19) Weingand K., Brown G., Hall R. et al. (1996). Harmonization
of Animal Clinical Pathology Testing in Toxicity and Safety Studies.
Fundam. andAppl. Toxicol. 29:198-201.
(20) World Health Organization (WHO). Guidelines for Evaluation
of Drugs for Use in Man. WHO Technical Report Series No. 563.
(21) World Health Organization (WHO). Part I. Environmental Health
Criteria 6, Principles and Methods for Evaluating the Toxicity of Chemi-
cals. WHO, Geneva (1978).
(22) 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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