United States Prevention, Pesticides EPA712-C-99-352
Environmental Protection and Toxic Substances July 1999
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.8355
Combined Chronic
Toxicity/Carcinogenicity
Testing of Respirable
Fibrous Particles
"Public Draft'
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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 EPA's
World Wide Web site (http://www.epa.gov/epahome/research.htm) under
the heading "Researchers and Scientists/Test Methods and Guidelines/
OPPTS Harmonized Test Guidelines" 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 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 870.8355 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, and EPA-748-R-96-001 Workshop Report on Chronic In-
halation Toxicity and Carcinogenicity Testing of Respirable Fibrous Par-
ticles.
(b) Purpose. The objective of a combined chronic toxicity/carcino-
genicity study is to determine the effects of a fibrous substance in a mam-
malian species following prolonged and repeated inhalation exposure. The
application of this guideline should generate data which identify the major-
ity of chronic and carcinogenicity effects and determine dose-response re-
lationships. The design and conduct should allow for the detection of neo-
plastic effects and a determination of the carcinogenic potential as well
as general toxicity, including neurological, physiological, biochemical, and
hematological effects and exposure-related morphological (pathology) ef-
fects.
(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.
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 in susceptible tissues.
Dose/concentration in a combined chronic toxicity/carcinogenicity
study is the amount of test substance administered via inhalation routes
for a period of up to 24 months. Concentration of fibrous particles is ex-
pressed as absolute number of fibers per cubic centimeter (f/cc).
Gravimetric concentration 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).
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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-effect-level (NOEL) is the maximum dose 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 is respirable
by humans. (Aerodynamic diameter, the most important determinant of
the respirability of a fiber, is different from its actual, geometry 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 (im).
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 has been demonstrated to be the most appropriate species because of
its susceptibility to fiber-induced lung diseases (fibrosis and lung tumors).
Commonly used laboratory strains should be employed. The strain selected
should be susceptible to the carcinogenic or toxic effect of fibrous par-
ticles. The criteria for a suitable strain include:
(A) A low background rate of neoplasia.
(B) A low background rate of pulmonary disease.
(C) Longevity.
(D) A history of laboratory use.
When the fiber in question is expected to be a mesothelioma inducer
(such as erionite fiber), testing in the hamster as a second rodent species
is recommended since the hamster appears to be more sensitive than the
rat with respect to fiber-induced mesotheliomas. 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.
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(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 dose level and concurrent control group. At least
40 additional rodents (20 males and 20 females) should be used for sat-
ellite dose groups and the satellite control group. The purpose of the sat-
ellite groups are for interim sacrifices for lung burden analysis, BALF
(bronchoalveolar lavage fluid) analysis and the evaluation of pathology
other than neoplasm (e.g., fibrosis).
(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. Sur-
vival in any group should not fall below 25 percent at 24 months.
(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.
(C) The relative humidity of the experimental animal rooms should
be 30 to 70 percent.
(D) Where lighting is artificial, the sequence should be 12 h light/
12 h 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.
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(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, size distribution, aero-
dynamic diameter, chemistry, density, solubility, surface characteristics,
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 impuri-
ties.
(ii) To maximize sensitivity of animal inhalation exposure studies to
health effects of fibers, the test material should consist of rat-respirable
fibers and should be enriched with the most potent human respirable frac-
tion (i.e., long, thin fibers); therefore, rodent inhalation exposure studies
should use an exposure aerosol that is, as far as is technically feasible,
enriched with the following fiber size fractions: Rat-respirable fibers with
aspect ratio of at least 3:1 and aerodynamic diameter less than 3 (im, and
human respirable fibers with lengths of at least 20 (im or fibers with high
aspect ratios. The fraction of long fibers (>20 (im) should be specified;
10 percent to 20 percent would be appropriate. 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.
(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 fiber concentration to be tested in a chronic 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). The MAC should be considered based on a
combination of the following parameters determined during lung burden
analysis and BALF (bronchoalveolar lavage fluid) analysis in a 90-day
subchronic inhalation study: Altered alveolar macrophage mediated par-
ticle clearance rate, fiber lung burden normalized to exposure concentra-
tion, cell proliferation, histopathology, inflammation (quantitatively deter-
mined as percentage increase in polymorphonuclear leukocytes [PMNs]
in lung lavage samples) and lung weight. An appropriate lung burden of
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critical fibers (long and thin) should be achieved. In addition, impairment
of clearance should be assessed in a 90-day subchronic inhalation study
via challenge with a tagged particle. Clearance should be assessed after
the 90-day exposure period and the clearance of the labelled particles
should be measured over a recovery period for another 3 months. These
parameters should be considered together, rather than individually, in an
attempt to define a MAC.
(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.
(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.
(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.
(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 surround-
ing 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
30 to 70 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.
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(vii) During the development of the generating system, fiber/particle
size analysis should be performed to establish the stability of aerosol con-
centrations with respect to fiber size. During exposure, analysis should
be conducted to determine 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 to provide data on biopersist-
ence 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. 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.
(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); 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 once
each day for morbidity and mortality. Appropriate actions should be taken
to minimize loss of animals from the study (e.g., necropsy or refrigeration
of those animals found dead and isolation or sacrifice of weak or moribund
animals).
(ii) A careful clinical examination should be made at least once week-
ly. Observations should be detailed and carefully recorded, preferably
using explicitly defined scales. Observations should include, but not be
limited to, evaluation of skin and fur, eyes and mucous membranes, res-
piratory and circulatory effects, autonomic effects such as salivation,
central nervous system effects, including tremors and convulsions, changes
in the level of motor activity, gait and posture, reactivity to handling or
sensory stimuli, grip strength and stereotypies or bizarre behavior (e.g.,
self-mutilation, walking backwards).
(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.
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(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. 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 conduct of the study and at termination. 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. If clinical observations suggest deterioration
in health of the animals during the study, a differential blood count of
the affected animals should be performed.
(i) Hematology. The recommended parameters are: Hemoglobin and
hematocrit concentrations, red blood cell count, white blood cell count,
differential leukocyte count, platelet count and a measure of clotting poten-
tial, such as prothrombin time or thromboplastin time.
(ii) Clinical chemistry. Parameters which are considered appropriate
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 clinical
toxicity. Suggested blood clinical chemistry determinations are:
(A) Electrolytes.
(7) Calcium.
(2) Chloride.
(3) Magnesium.
(4) Phosphorous.
(5) Potassium.
(6) Sodium.
(B) Enzymes.
(7) Alkaline phosphatase.
(2) Alanine aminotransferase.
(3) Aspartate aminotransferase.
(4) Gamma glutamyl transferase.
(C) Other.
(7) Albumin.
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(2) Blood creatinine.
(3) Blood urea nitrogen.
(4) Globulin.
(5) Glucose (fasting).
(6) Total Bilirubin.
(7) Total cholesterol.
(8) Total serum protein.
Other determinations which may be necessary for an adequate toxi-
cological evaluation include analyses of lipids, hormones, acid/base bal-
ance, methemoglobin and cholinesterase. Additional clinical chemistry
may be employed where necessary to extend the investigation of observed
effects.
(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), protein, glucose, ketones, bilirubin,
occult blood (semiquantitatively), and microscopy of sediment
(semiquantitatively).
(9) BALF (bronchoalveolar lavage fluid). BALF analysis should be
conducted at various time points (e.g., at 3, 6, 12 , 18 and 24 month)
on subgroups of 5-6 rats/group. Lavage parameters to be determined in-
clude: Total cell count, differential cell counts (PMN, alveolar
macrophages, lymphocytes, and others), total protein, LDH and beta-glucu-
ronidase as examples of cytoplasmic and lysosomal enzymes.
(10) 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.
(11) 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) The following organs and tissues, or representative samples there-
of, should be preserved in a suitable medium for possible future
histopathological examination.
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(A) Digestive system.
(7) Salivary glands.
(2) Esophagus.
(3) Stomach.
(4) Duodenum.
(5) Jejunum.
(6) Ileum.
(7) Cecum.
(8) Colon.
(9} Rectum.
(10} Liver.
(11} Pancreas.
(12} Gallbladder (mice).
(13} Bile duct (rat).
(B) Nervous system.
(1} Brain (multiple sections).
(2) Pituitary.
(3} Peripheral nerves.
(4) Spinal cord (three levels).
(5) Eyes (retina, optic nerve).
(C) Glandular system.
(1} Adrenals.
(2) Parathyroids.
(3} Thyroids.
(D) Respiratory system.
(1} Pleura.
(2)Trachea.
(3) Lung.
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(4) Pharynx.
(5) Larynx.
(6) Nose.
(E) Cardiovascular/hematopoietic system.
(7) Aorta (thoracic).
(2) Heart.
(3) Bone marrow.
(4) Lymph nodes.
(5) Spleen.
(6) Thymus.
(F) Urogenital system.
(7) Kidneys.
(2) Urinary bladder.
(3) Prostate.
(4) Testes/epididymides.
(5) Seminal vesicles.
(6} Uterus.
(7) Ovaries.
(G) Other.
(7) All gross lesions and masses.
(2) Sternum and/or femur.
(iv) 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.
(v) Inflation of lungs and urinary bladder with a fixative is the optimal
method for preservation of these tissues. The proper inflation and fixation
of the lungs in inhalation studies is essential for appropriate and valid
histopathological examination.
(vi) Information from clinical pathology and other in-life data should
be considered before microscopic examination, since these data may pro-
vide significant guidance to the pathologist.
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(12) Histopathology. (i) The following histopathology should be per-
formed:
(A) Full histopathology on the organs and tissues, listed under para-
graph (d)(ll)(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/tissues in all animals. Organs/tissues of the res-
piratory tract represent 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.
(D) Livers and kidneys of 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
(d)(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. Tissues should be trimmed to a maximum thickness of 0.4
cm for processing.
(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.
(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
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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. In order to detect a positive
effect, the power of the study should be such that the type I error is con-
trolled at 0.05 and as the type II error is controlled at 0.2.
(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, the following
specific information should be reported:
(A) Test substance characterization should include:
(7) Chemical identification.
(2) Lot or batch number.
(3) Physicochemical properties (i.e., fiber morphology, dimension,
size distribution, aerodynamic diameter, chemistry, density, solubility, sur-
face characteristics, the ability of a fiber to split longitudinally or cross-
sectionally).
(4) Purity/impurities.
(B) Test system should contain data on:
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(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) Dose 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.
Actual (analytical or gravimetric) concentration in the
breathingzone.
(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).
(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.
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(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 performed.
(G) Results of clinical chemistry tests performed.
(H) Results of urinalysis tests performed.
(I) Results of lung burden analysis.
(J) Results of BALF (bronchoalveolar lavage fluid) analysis.
(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.
(f) Quality assurance. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment. The study must be conducted in compliance with the GLP reg-
ulations as described by the Agency (40 CFR parts 160 and 792) and
the OECD Principles of GLP (ISBN 92-64-12367-9).
(g) References. The following references should be consulted for ad-
ditional background information on this guideline.
(1) Dement, J.M. Overview: Workshop on Fiber Toxicology Research
Needs. Environmental Health Perspective 88:261-268 (1990).
(2) Harington, J.S. Fiber Carcinogenesis: Epidemiologic Observations
and the Stanton Hypothesis. J. National Cancer Institute 67:977-987
(1981).
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(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-
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20:S 1-8222(1994).
(5) Leong, 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).
(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 Phar-
macology 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. Fund. Appl. Toxicolo.
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) 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)
(11) 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).
(12) Sontag, J.M., Page, N.P. and Saffiotti, U. Guidelines for Carcino-
gen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United States
Cancer Institute, Division of Cancer Control and Prevention, Carcino-
genesis Bioassay Program.
(13) United States Environmental Protection Agency. Health Effects
Test Guidelines—Combined Chronic Toxicity/Oncogenicity. 40 CFR Part
798.3320 FEDERAL REGISTER, pp. 165-172, July 1, 1998.
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(14) 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.
(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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