United States Prevention, Pesticides EPA 712-C-01 -352
Environmental Protection and Toxic Substances July 2001
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.8355
Combined Chronic
Toxicity/Carcinogenicity
Testing of Respirable
Fibrous Particles
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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. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Internet Home Page at http://www.epa.gov/opptsfrs/home/
guidelin.htm or in paper by contacting the OPP Public Docket at (703)
305-5805 or by e-mail: opp-docket@epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: oppdocket@epa.gov.
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 the EPA Internet Home Page
at http://www.epa.gov/opptsfrs/home/guidelin.htm.
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OPPTS 870.8355 Combined Chronic Toxicity/Carcinogenicity Testing
of Respirable Fibrous Particles.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Toxic Substances Control Act (TSCA) (15 U.S.C.
2601).
(2) Background. The source materials used in developing this
OPPTS test guideline are 40 CFR 798.3320 Combined Chronic Toxicity/
Oncogenicity, EPA-748-R-96-001 Workshop Report on Chronic Inhala-
tion Toxicity and Carcinogenicity Testing of Respirable Fibrous Particles,
and EPA's FIFRA Scientific Advisory Panel Report (Report No. 2000-
OX, 01/05/2001).
(b) Purpose. The objective of a combined chronic toxicity/carcino-
genicity study is to determine the effects of a fibrous substance in at least
a rodent species following prolonged and repeated inhalation exposure.
The fibrous substances to be tested under this guideline will be selected
based on data from appropriate short-term screening tests indicative of po-
tential health hazard and risk concern. The application of this guideline
should generate data which identify the majority of chronic toxic and car-
cinogenic effects and determine dose-response relationships. The design
and conduct should allow for the detection of neoplastic and non-neo-
plastic effects of the target tissues. In addition, it may also determine gen-
eral toxicity and exposure-related morphological (pathology) effects if the
fiber substance under test is known to have such toxic potential (e.g., based
on results of pre-chronic studies/analyses). While the guideline will have
application to testing of organic fibers, additional considerations may be
necessary for study of organic fibers.
(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 the test sub-
stance by the inhalation route of exposure.
Chronic toxicity is the adverse effects occurring as a result of the
repeated daily exposure of experimental animals to the test substance by
the inhalation route of exposure.
Concentration in a combined chronic toxicity/carcinogenicity study
of fibrous particles is the amount of test substance administered via inhala-
tion routes for a period of up to 24 months. Concentration of fibrous par-
ticles is expressed as absolute number of fibers per cubic centimeter (f/
cc). Exposure concentrations should also be expressed by fiber length, e.g.,
World Health Organization (WHO) fibers (greater than 5 (im in length)/
cc, fibers with length greater than 10, 15 and 20 (im/cc. Gravimetric con-
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centration expressed as milligrams per cubic meter (mg/m3) is used for
daily monitoring of the generated aerosols in order to achieve the intended
number of fiber per unit of aerosol volume (f/cc).
Dose in a combined chronic toxicity/carcinogenicity study of fibrous
particles is the amount of test substance deposited upon inhalation or the
amount of the test substance retained in the lung after certain time post-
exposure. Dose of fibrous particles is expressed as number of fibers per
lung. Fiber number is expressed by total fibers and by fiber length, e.g.,
WHO fibers (larger than 5 (im in length), fibers larger than 10, 15 and
20 (im in length.
Fibrous particles/fibers are generally defined as elongated particles
with a length-to-diameter ratio (i.e., aspect ratio) equal to or greater than
3 to 1. This definition is presumed to include particles with varying shapes
such as rod-like, curly, or acicular (needle-like) shapes, and having dif-
ferent structural units commonly referred to as fibers, fibrils, or whiskers.
No-observed-adverse-effect-level (NOAEL) is the maximum con-
centration used in a study which produces no observed adverse effects.
Respirable means that the particle in question can penetrate to the
alveolar region upon inhalation. There are considerable differences in fiber
respirability between laboratory rodents and humans. A "rat-respirable
fiber'' is defined as a fiber having an aerodynamic diameter of less than
3 (im. A fiber having an aerodynamic diameter of less than 5 (im is res-
pirable by humans. (Aerodynamic diameter, the most important deter-
minant of the respirability of a fiber, is different from its actual, geometric
diameter. The aerodynamic diameter of a fiber is dependent on its density
and aspect ratio. For example, fibers having actual diameters of 0.25 to
2.0 (im would have aerodynamic diameters three to four times their actual
diameters if their density is lg/cm3 and their lengths are between 10 and
150 urn).
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance.
(d) Test procedure—(1) Animal selection—(i) Species and strain.
For the study of respirable fibrous particles via the inhalation route, the
rat should be the first rodent species used because of its susceptibility
to fiber-induced diseases such as pulmonary fibrosis, lung neoplasms and
mesothelioma. Commonly used laboratory strains should be employed. The
strain selected should be susceptible to the carcinogenic or toxic effect
of fibrous particles. The criteria for a suitable strain include:
(A) A low background rate of neoplasia.
(B) A low background rate of pulmonary disease.
(C) Longevity.
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(D) A history of laboratory use.
Since the hamster appears to be more sensitive than the rat with re-
spect to fiber-induced mesothelioma, the hamster should be considered as
a second species when results of the rat study show pleural toxicity or
neoplasms and dose response data are needed for risk assessment purposes.
If other species are used, the tester should provide justification/reasoning
for the selection.
(ii) Age/weight. (A) Testing should be started with young healthy
animals as soon as possible after weaning and acclimatization.
(B) Dosing should generally begin no later than 8 weeks of age.
(C) At commencement of the study, the weight variation of animals
used should not exceed 20 percent of the mean weight for each sex.
(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 concentration level and concurrent control group.
At least 40 additional rodents (20 males and 20 females) should be used
for satellite dose groups and the satellite control group. The purpose of
the satellite groups are for interim sacrifices for lung burden analysis,
bronchoalveolar lavage fluid (BALF) analysis and the evaluation of pathol-
ogy other than neoplasm (e.g., fibrosis). Additional rodents (five to six
per groups) may also be needed for recovery groups to evaluate fiber per-
sistence and the progression or regression of a given lesion in the absence
of fiber exposure.
(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 18 months for
rats and 15 months for hamsters. Survival in any group should not fall
below 25 percent at 24 months for rats and 18 months for hamsters.
(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 should be housed individually during
exposure in inhalation studies.
(B) The temperature of the experimental animal rooms should be at
22 + 3°C.
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(C) The relative humidity of the experimental animal rooms should
be 50 + 20 percent.
(D) The daily light cycle should be maintained at 12 h light and 12
h dark, whether artificial or natural.
(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.
(2) Control and test substances, (i) One lot of the test substance
should be used throughout the duration 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 charac-
terization of the test substance, including the purity and physicochemical
properties (e.g., fiber morphology, dimension, bivariate size distribution,
aerodynamic diameter, chemistry, density, dissolution rate, surface charac-
teristics, the ability of a fiber to split longitudinally or cross-sectionally)
of the test fiber, and, if possible, the name and quantities of contaminants
and impurities.
(ii) To maximize sensitivity of animal inhalation exposure studies to
health effects of fibers, the test material should consist of rat-respirable
fibers which should be enriched with the most potent fraction of long,
thin fibers or fibers with high aspect ratios. As far as is technically fea-
sible, the aerosol should be cleaned up from non-fibrous particles. The
aerosol should be characterized in term of fiber and non-fiber/particle size
and number; fiber number should be expressed by total fibers and by fiber
length, e.g., WHO fibers (greater than 5 (im in length), fibers greater than
10, 15 and 20 (im in length. If enriching the test aerosol with long, thin
fibers is not feasible, the reasons should be clearly stated and justified,
and the enrichment should be for the longest fibers or fibers with the high-
est aspect ratios available. The aerosolized fibers should be discharged to
Boltzmann equilibrium before being delivered to the test species.
(3) Control groups. A concurrent control group (50 males and 50
females) and a satellite control group (10 males and 10 females) are re-
quired. These groups should be untreated. Animals in the satellite control
group should be sacrificed at the same time the satellite test group is termi-
nated. A positive control group may not need to be included in every
study, but each new test system (including use of a different animal species
and strain) should be validated with a positive control material.
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(4) Concentration levels selection, (i) For dose-response relation-
ships analysis, at least three concentration levels should be used, in addi-
tion to the concurrent control group. Along with other information (de-
crease in body weight, systemic toxicity etc.), data should be obtained on
lung burden analysis and BALF analysis in a 90-day subchronic inhalation
study to assist in establishing the chronic exposure concentration levels.
A combination of the following parameters should be evaluated: Altered
alveolar macrophage mediated particle clearance rate, fiber lung burden
normalized to exposure concentration, cell proliferation, histopathology,
inflammation (marker enzyme activities, total protein content, total cell
count, cell differential, and cell viability in lung lavage samples) and lung
weight. In addition, impairment of clearance should be assessed in a 90-
day inhalation study via challenge with a tagged particle; clearance should
be assessed after the 90-day exposure period and at a recovery period for
another 3 months. Exposure concentration levels should be spaced to
produce a gradation of effects. A rationale for the concentrations selected
must be provided.
(ii) The highest fiber concentration to be tested in a chronic inhalation
study is known as the maximum aerosol concentration or MAC. The MAC
should be based on the total number of inhaled particles (fibers and non-
fibrous particles combined); an appropriate lung burden of critical fibers
(long and thin) should be achieved. The MAC should be set at a level
at which some degree of impaired clearance and toxicity are observed.
All the parameters in the 90-day studies should be considered together,
rather than individually, in an attempt to define a MAC, and should be
presented to the agency for evaluation before the chronic study is con-
ducted.
(iii) The intermediate concentration levels should be spaced to
produce a gradation of toxic effects.
(iv) The lowest concentration level should produce minimal or no
evidence of toxicity.
(5) Administration of the test substance. Inhalation is the major
route of human exposure of fibrous particles, and chronic inhalation stud-
ies in rodents are deemed appropriate tests for evaluating inhalation hazard
and risk of fibers to humans. Either nose-only or whole-body exposure
can be used. If whole-body exposure is used, validation of sufficient fibers
reaching the gas-exchange region of the lungs must be provided.
(i) 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 24 months in rats.
However, based primarily on practical considerations, exposure for 6 h/
day on a 5-day per week basis is acceptable. Due to snorter life span
of hamsters, their exposure duration could be shorter, based upon survival/
lifetime expectancy.
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(ii) 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 sur-
rounding areas.
(iii) 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.
The animals should be acclimated and heat stress minimized.
(iv) The temperature at which the test is performed should be main-
tained at 22 + 3°C. The relative humidity should be maintained between
50 + 20 percent.
(v) The rate of air flow should be monitored continuously but re-
corded at least every 30 minutes.
(vi) Temperature and humidity should be monitored continuously but
should be recorded at least every 30 minutes.
(vii) The system used to generate fibrous aerosols must not cause
significant breakage and contamination of the test substance. During the
development of the generating system, fiber/particle size analysis should
be performed to establish the stability of aerosol concentrations with re-
spect to fiber size. During exposure, analysis should be conducted to deter-
mine the consistency of fiber size distribution and the actual concentrations
of the test substance. The frequency of exposure atmosphere monitoring
should be daily for mass concentration, weekly for fiber concentration and
bivariate size distribution.
(viii) The actual concentrations of the test substance should be meas-
ured in the breathing zone. Lung burden analyses should be conducted
after 3, 6, 12, 18, and 24 months of exposure in the rat and after 3, 6,
12, and 18 months in the hamster to provide data on biopersistence of
the test fibers and serve as a better measure of internal dose. Data also
should be obtained on fiber deposition in the nasal cavity and the fiber
burden in the thoracic lymph nodes. The fibers should be analyzed for
number, bivariate size distribution and chemistry. Fiber number should be
expressed by total fibers and by fiber length, e.g., WHO fibers (greater
than 5 (im in length), fibers greater than 10, 15 and 20 (im in length.
For fiber burden analysis, one of the two lungs (left or right) should be
used, rather than only the accessory lobe. Five to six animals per exposure
group should be studied at each time point. Lung burden and fiber size
distribution should be reported as number of fibers per gram of dry lung
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tissue, as well as number of fibers per gram of wet lung tissue. The burden
should be extrapolated to the whole lung. The method for lung burden
analysis ("lung digestion") must be validated. It is recommended to in-
clude "recovery" groups of animals from exposure at 3, 6, 12, and 18
months and then hold until 24 months for evaluation.
(ix) Feed should be withheld during exposure. Water may also be
withheld during exposure.
(6) Observation period. The chronic inhalation exposure study with
fibers should be a lifetime study. The animals should be observed for their
life span after the exposure duration is completed (at least 24 months for
rats and 18 months for hamsters); final sacrifice should be carried out
only when survival of the control group reaches 20 percent.
(7) Observation of animals, (i) Observations should be at least twice
each day for morbidity and mortality. Appropriate actions should be taken
to minimize loss of animals from the study (e.g., necropsy or refrigeration
of those animals found dead and isolation or sacrifice of weak or moribund
animals). General clinical observations should be made at least once a day.
(ii) A careful clinical examination should be made at least once week-
ly. Observations should be detailed and carefully recorded, preferably
using explicitly defined scales. Observations should include evaluation of
skin and fur, eyes and mucous membranes, respiratory and circulatory ef-
fects, autonomic effects such as salivation, central nervous system effects,
including tremors and convulsions, changes in the level of motor activity,
gait and posture, reactivity to handling or sensory stimuli, grip strength
and stereotypies or bizarre behavior (e.g., self-mutilation, walking
backwards) if the fiber substance under test is known to have such toxic
potential (e.g., based on data of pre-chronic studies).
(iii) Body weights should be recorded individually for all animals:
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 fre-
quent weighing to facilitate monitoring of health status.
(iv) 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.
(8) Clinical pathology. If hematological and biochemical effects are
seen in the subchronic study, hematology, clinical chemistry and urinalyses
should be performed from 10 animals per sex per group 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. Over-
night fasting of animals prior to blood sampling is recommended.
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(i) Hematology. The recommended parameters are: Hemoglobin and
hematocrit concentrations, red blood cell count, 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
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 clin-
ical 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 alkaline
phosphatase, alanine aminotransferase, aspartate aminotransferase, sorbitol
dehydrogenase, or gamma glutamyl transferase.) should also be measured.
(iii) Urinalyses. The following determinations should be made from
either individual animals or on a pooled sample per sex per group: Appear-
ance (volume and specific gravity), pH, protein, glucose, ketones, bilirubin,
occult blood (semiquantitatively), and microscopy of sediment
(semiquantitatively).
(9) Bronchoalveolar lavage fluid (BALF) analysis. BALF analysis
should be conducted at the time of the 12 and 24 month sacrifices on sub-
groups of 5 rats/group; additional time points are optional. Lavage param-
eters to be determined should include: total cell count, differential cell
counts (polymorphonuclear leukocytes (PMN), alveolar macrophages,
lymphocytes, and others), total protein, lactate dehydrogenase and (3-glucu-
ronidase as examples of cytoplasmic and lysosomal enzymes. It is rec-
ommended to include ' 'recovery'' groups of animals from exposure at the
interim time points and then hold until 24 months for evaluation.
(10) Lung clearance. It is recommended that animals be tested for
impaired lung clearance for a pulse of a small spherical particle at 9 and
18 months and recovery of the animals be followed with sacrifices at the
same intervals as the animals exposed for 24 months.
(11) Ophthalmological examination. If changes in eyes are detected
in the subchronic study, examinations of the eyes should be made on all
animals using an ophthalmoscope or an equivalent device prior to the ad-
ministration 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 in the high-dose groups, all animals should be examined.
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(12) 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) The liver, lungs, kidneys, brain, spleen, and gonads should be
trimmed and weighed wet, as soon as possible after dissection to avoid
drying. 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) In inhalation studies of fibers, the entire respiratory tract, includ-
ing nose, pharynx, larynx, paranasal sinuses, lungs, trachea and pleura
should be examined and preserved. In addition, since fiber detention may
occur at the rib cage and diaphragm, these organ tissues should also be
examined and preserved.
(iv) Inflation of lungs with a fixative is the optimal method for preser-
vation of these tissues. The proper inflation and fixation of the lungs in
inhalation studies is essential for appropriate and valid histopathological
examination. It is recommended the lungs from the scheduled sacrifices
be inflated with an appropriate fixative to allow the grading of lesions
and better evaluation of subtle changes.
(v) 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, duodenum,
jejunum, illeum, cecum, colon, rectum, liver, pancreas, gallbladder (when
present).
(B) Nervous system brain (multiple sections), pituitary, peripheral
nerves, spinal cord (three levels), eyes (retina, optic nerve).
(C) Glandular system adrenals, parathyroids, thyroid.
(D) Cardiovascular/hematopoietic system aorta (thoracic), heart, bone
marrow, lymph nodes, spleen, thymus.
(E) Urogenital system kidneys, urinary bladder, prostate, testes/
epididymides, seminal vesicles, uterus, ovaries, female mammary gland.
(F) Other all gross lesions and masses, skin.
(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.
(13) Histopathology. (i) The following histopathology should be per-
formed in all animals:
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(A) Target organs/tissues. Organs/tissues of the respiratory tract rep-
resent the target organs/tissues for evaluating effects of inhaled fibers.
Major effects include pulmonary fibrosis, lung tumors and mesotheliomas.
Special attention to examination of the lungs of rodents should be made
for evidence of infection since this provides an assessment of the state
of health of the animals.
(B) The rib cage and diaphragm. The rib cage and diaphragm should
be examined histopathologically for mesothelial lesions. The section of
diaphragm for histology should be cut in a manner to show the muscular
as well as the non-muscular portion.
(ii) (A) If gross lesions are observed on the organs and tissues listed
under paragraph (d)(12)(ii) or paragraph (d)(12)(v) of this guideline, full
histopathology should be performed on all animals in the control and high
dose groups and of all animals that died or were killed during the study.
(B) 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 on all animals including those
which died during the experiment or were killed in a moribund condition.
(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. The histology slides from the scheduled sacrifices should,
in addition to standard hematoxylin and eosin, be stained with a method
that identifies collagen (fibrosis).
(v) Histopathological evaluation should incorporate both qualitative
description of lesions and rigorous quantitation. A consistent approach
should be taken to record and grade the findings of the lesions using a
standardized method in contemporary fiber studies. "Image analysis" may
be used to quantify the severity of lesions in the lungs.
(e) Data and reporting—(1) Treatment of results, (i) Data should
be summarized in tabular form, showing for each test group the number
of animals at the start of the test, the number of animals showing lesions,
the types of lesions and the percentage of animals displaying each type
of lesion.
(ii) All observed results (quantitative and qualitative), should be eval-
uated by an appropriate statistical method. Any generally accepted statis-
tical 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, (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-
fects.
(ii) Non-neoplastic and neoplastic endpoints recorded should include,
but not be limited to, epithelial hyperplasia, alveolar bronchiolization,
metaplasia, adenomas, mesotheliomas, and carcinomas. A dissecting mi-
croscope should be used to examine for mesotheliomas. In distinguishing
between hyperplasia and mesothelioma, standard diagnostic criteria should
be applied to identified lesions. Established published guidelines on the
use of blinding in histopathology should be followed, e.g., those published
by the Society of American Pathologists.
(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 hamsters and 18 months
for rats. Survival should not fall below 25 percent at 18 months for ham-
sters and 24 months for rats. For acceptance of the results of a chronic
inhalation exposure study with fibers as negative, the study must have been
designed and conducted according to the criteria outlined previously, the
health effects of concern must not be significantly more frequent in the
exposure groups than in the control group. Sample sizes for toxicity studies
should be large enough to detect a specific effect level (e.g., a 10% change
in fibrosis or tumor) with a = P (Type I error) = 0.05 and power = 1-
P (Type II error) > 0.80.
(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 and 40 CFR part 160, the following
specific information should be reported:
(A) Test substance characterization should include:
(7) Chemical identification.
(2) Lot or batch number.
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(3) Physicochemical properties (i.e., fiber morphology, dimension,
size distribution, aerodynamic diameter, chemistry, density, dissolution
rate (Kdis), surface characteristics, the ability of a fiber to split longitu-
dinally or cross-sectionally).
(4) Purity/impurities.
(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.
(C) Test procedure should include the following data:
(7) Method of randomization used.
(2) Full description of experimental design and procedure.
(3) Concentration regimen including levels, methods, and volume.
(D) 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 fiber/
particulate aerosol concentrations and size should be described.
(E) 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) Fiber and particle size distribution, and calculated mass median
aerodynamic diameter (MMAD) and geometric standard deviation (GSD).
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Fiber number reported should also be expressed by fiber length, e.g., WHO
fibers (>5 (im in length), fibers >10, >15, >20 (im in length.
(6) Explanation as to why the desired chamber concentration and/
or fiber size could not be achieved (if applicable) and the efforts taken
to comply with this aspect of the guidelines.
(F) Test results—(1) Group animal data. Tabulation of toxic re-
sponse 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.
(2) 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) Results of ophthalmological examination, when performed.
(F) Results of hematological tests, when performed.
(G) Results of clinical chemistry tests, when performed.
(H) Results of urinalysis tests, when performed.
(I) Results of lung burden analysis.
(J) Results of BALF analysis.
(K) Necropsy findings including absolute/relative organ weight data.
(L) Detailed description of all histopathological findings.
Histopathological evaluation should incorporate both qualitative descrip-
tion of lesions and rigorous quantitation.
(M) Statistical treatment of results where appropriate.
(N) Historical control data.
(f) Quality assurance. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
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equipment. The study must be conducted in compliance with the GLP reg-
ulations as described by the Agency (40 CFR parts 160 and 792) and
the OECD Principles of GLP (ISBN 92-64-12367-9).
(g) References. The following references should be consulted for ad-
ditional background information on this guideline.
(1) Dement, J.M. Overview: Workshop on Fiber Toxicology Research
Needs. Environmental Health Perspectives 88:261-268 (1990).
(2) Harington, J.S. Fiber Carcinogenesis: Epidemiologic Observations
and the Stanton Hypothesis. Journal of National Cancer Institute 67:977-
987(1981).
(3) International Union Against Cancer. Carcinogenicity Testing:
UICC Technical Report Series, Vol.2, Ed. I Berenblum. International
Union Against Cancer, Geneva (1969).
(4) ISTRP. International society of Regulatory Toxicology and Phar-
macology. Proceedings Symposium on Synthetic Vitreous Fibers: Sci-
entific and Public Policy Issues. Regulatory Toxicology and Pharmacology
20:S 1-8222(1994).
(5) Lewis, T.R., Morrow, P.E., McClellan, R.O., Raabe, O.G., Ken-
nedy, G.L., Chhabra, R.S., Schwetz, B.A., Goehl, T.J., and Roycroft, J.H.
Establishing aerosol exposure concentrations for inhalation toxicity studies.
Toxicology and Applied Pharmacology 99:377-383 (1989).
(6) McClellan, R.O., Miller, F.J., Hestersberg, T.H., Warheit, D.B.,
Bunn, W.B., Kane, A.B., Lippmann, M., Mast, R.W., McConnell, E.E.
and Reinhardt, C.F. Approaches to Evaluating the Toxicity and Carcino-
genicity of Man-Made Fibers: Summary of a workshop Held November
11-13, 1991, Durham, North Carolina. Regulatory Toxicology and Pharma-
cology 16:321-364 (1992).
(7) Morrow, P.E., Haseman, J.K., Hobbs, C.H., Driscoll, K.E., Vu,
V., and Oberdorster, G. Workshop overview: The maximum tolerated dose
for inhalation bioassays: toxicity vs. overload. Fundamental Applied Toxi-
cology 29:155-167 (1996).
(8) Organization for Economic Cooperation and Development. Guide-
lines for Testing of Chemicals, Section 4-Health Effects, Part 453 Com-
bined Chronic Toxicity/Carcinogenicity Studies, Paris (1981).
(9) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines.
Journal of Environmental Pathology and Toxicology 11:161-182 (1977).
(10) Spurny, K.R., Stober, W., Opiela, H. and Weiss, G. Size-selec-
tive Preparation of Inorganic Fibers for Biological Experiments. American
Industrial Hygiene Association Journal 40:20-37 (1979).
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(11) Sontag, J.M., Page, N.P. and Saffiotti, U. Guidelines for Car-
cinogen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United
States Cancer Institute, Division of Cancer Control and Prevention, Car-
cinogenesis Bioassay Program.
(12) United States Environmental Protection Agency. Health Effects
Test Guidelines: Combined Chronic Toxicity/Oncogenicity. 40 CFR
798.3320 pp. 165-172.
(13) United States Environmental Protection Agency. Office of Pollu-
tion Prevention and Toxics. Workshop on Chronic Inhalation Toxicity and
Carcinogenicity Testing of Respirable Fibrous Partcles. EPA-748-R-96-
001, January 1996.
(14) United States Environmental Protection Agency. FIFRA Sci-
entific Advisory Panel Meeting on Test Guidelines for Chronic Inhalation
Toxicity and Carcinogenicity of Fibrous Partcles, September 26, 2000.
SAP Report No. 2000-OX, 01/05/2001.
(15) Vu, V., Barrett, J.C., Roycroft, J., Schuman, L., Dankovic, D.,
Baron, P., Martonen, T., Pepelko, W. and Lai, D. Workshop Report:
Chronic Inhalation Toxicity and Carcinogenicity Testing of Respirable Fi-
brous Particles. Regulatory Toxicology and Pharmacology 24:202-212
(1996).
(16) World Health Organization (WHO). Part I. Environmental Health
Criteria 6, Principles and Methods for Evaluating the Toxicity of Chemi-
cals. WHO, Geneva. (1978).
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