United States       Prevention, Pesticides     EPA712-C-99-352
          Environmental Protection    and Toxic Substances     July 1999
          Agency         (7101)
&EPA    Health Effects Test
          OPPTS 870.8355
          Combined Chronic
          Testing of Respirable
          Fibrous Particles
                "Public Draft'

     This guideline is one  of a series of test guidelines  that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency  for review under Federal regulations.

     The Office of Prevention,  Pesticides and Toxic Substances (OPPTS)
has  developed this guideline through  a  process of harmonization that
blended the testing  guidance and requirements that existed in the Office
of Pollution Prevention and Toxics  (OPPT) and appeared in Title 40,
Chapter I,  Subchapter R of the Code of Federal Regulations  (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical  Information Service (NTIS) and  the guidelines pub-
lished by the Organization  for Economic Cooperation and Development

     The purpose of harmonizing these guidelines into a single set of
OPPTS  guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency  under the Toxic  Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide,  Fungicide and Rodenticide Act
(7U.S.C. 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

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.

     (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-

     (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-

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

     Carcinogenicity is the development of neoplastic lesions as a result
of the repeated  daily exposure of experimental animals to 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).

     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.

     (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.

     (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-

     (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

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.

     (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

     (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.

     (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.

     (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

     (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

     (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.


(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.
(3) Lung.

     (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.

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

     (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


clinical abnormalities), gross lesions, identified target organs, body weight
changes, effects on mortality and any other general or specific toxic  ef-

     (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-

     (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) Dose regimen including levels, methods, and volume.

     (D) Test conditions. The following exposure conditions must be re-

     (7) Description of exposure apparatus including design, type, dimen-
sions, source of air, system for generating particulates and aerosols, meth-
od of conditioning air, treatment of exhaust air and the method of housing
the animals in a test chamber.

     (2) The equipment for measuring  temperature,  humidity, and  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

     (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.


     (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

     (B)  Time of observation of each abnormal sign and its  subsequent

     (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


     (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) 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.

     (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.


     (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

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