United States       Prevention, Pesticides     EPA712-C-98-204
           Environmental Protection    and Toxic Substances     August 1998
           Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.3465
           90-Day Inhalation

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

    The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through  a process of harmonization that
blended the testing  guidance  and requirements that  existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in title 40, chap-
ter I, subchapter R of the Code  of Federal Regulations (CFR), the Office
of Pesticide Programs (OPP) which appeared in publications of  the Na-
tional Technical Information Service (NTIS)  and the guidelines published
by the Organization for Economic Cooperation and Development (OECD).

    The purpose of harmonizing these  guidelines  into  a single set of
OPPTS  guidelines is to minimize variations among the testing procedures
that must be performed to meet the data  requirements of the U. S. Environ-
mental Protection Agency  under the Toxic  Substances  Control Act  (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).

    Final  Guideline Release: This guideline is available from the U.S.
Government Printing Office,  Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World  Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

OPPTS 870.3465   90-Day inhalation toxicity.
     (a) Scope—(1) Applicability. This guideline is intended to meet test-
ing  requirements   of  both  the  Federal  Insecticide,  Fungicide,  and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

     (2) Background. The source materials used  in developing this har-
monized OPPTS test  guideline  are 40 CFR 798.2450 Inhalation Toxicity;
OPP  82-4  90-Day  Inhalation—Rat  (Pesticide Assessment Guidelines,
Subdivision F—Hazard Evaluation; Human and Domestic  Animals) EPA
report 540/09-82-025,  1982; and OECD 413  Subchronic Inhalation Tox-
icity: 90-Day.

     (b) Purpose. In the assessment and evaluation of the toxic character-
istics of a gas,  volatile substance, or aerosol/particulate, determination of
subchronic inhalation toxicity may be carried  out  after initial information
on toxicity has been obtained by acute testing. The subchronic inhalation
study has been designed to permit the determination of the  no-observed-
effect-level  (NOEL) and toxic effects  associated  with  continuous or re-
peated exposure to a test substance for a  period  of 90  days. This  study
is not capable of determining those effects  that have a long latency period
for development (e.g., carcinogenicity and life shortening). Extrapolation
from the results of this study to humans is valid only to a limited degree.
It  can, however, provide useful information on health hazards likely to
arise from repeated exposures by the inhalation route over a limited period
of time. It will provide information on target  organs  and the possibilities
of accumulation, and can be of use in selecting concentration levels for
chronic studies and establishing safety criteria for human exposure. Haz-
ards of inhaled substances are  influenced by the inherent toxicity and by
physical factors such as volatility and particle size.

     (c) Definitions. The definitions in section 3  of the Toxic Substance
Control Act (TSCA) and the  definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.

     Aerodynamic  equivalent diameter is defined as the  diameter of a unit
density  sphere  having the same terminal  settling  velocity  as the  particle
in question, whatever its size, shape, and density.  It is used to predict
where in the respiratory tract such particles  may be  deposited.

     Concentration in a subchronic inhalation study is the  amount of test
substance administered via inhalation for a period  of 90 days. Concentra-
tion is expressed as weight of the test substance  per unit volume of air
(milligrams per liter or parts per million).

     Cumulative toxicity is the adverse effects of repeated concentration
occurring  as a  result of prolonged action  on, or  increased concentration
of the administered test substance or its metabolites in susceptible tissues.

    Inhalable diameter refers to that aerodynamic diameter of a particle
which is considered to be  inhalable for the organism. It is used to refer
to particles which are capable of being inhaled and may be  deposited any-
where within the respiratory tract

    No-observed-effect-level (NOEL) is the maximum concentration used
in a study which produces no adverse effects.

    Mass median aerodynamic diameter (MMAD) is the median aero-
dynamic diameter and along with the geometric standard deviation (GSD)
is used to describe the particle size distribution of any aerosol  statistically
based on the weight and size of the particles. Fifty percent  of the particles
by weight will be  smaller than the median diameter and 50 percent of
the particles will be larger.

    Subchronic inhalation toxicity is  the adverse effects  occurring as a
result of the repeated daily exposure of experimental animals to a chemical
by inhalation for part (approximately 10 percent) of a life span.

    (d) Limit  test. If exposure at a concentration of 1 mg/L (expected
human exposure may indicate the need for a higher  concentration), or
where this is not possible  due to  physical or chemical properties of the
test substance, the maximum attainable concentration produces no observ-
able toxic effects, then a full  study using three  concentrations might not
be necessary.

    (e) Test procedures—(1) Animal selection—(i)  Species and strain.
A mammalian species shall be used for testing. A variety of rodent species
may be used, although the rat is  the preferred  species. Commonly used
laboratory strains should be employed. If another mammalian species is
used, the tester shall provide justification/reasoning for its selection.

    (ii) Age/weight. (A) Testing should be started  with  young healthy
animals as soon as possible after weaning and acclimatization.

    (B) Dosing of rodents  should generally begin no later than 8-9 weeks
of age.

    (C) At the commencement of the study the weight variation of ani-
mals used  shall not exceed + 20 percent  of the  mean weight for each

    (iii) Sex. (A) Equal numbers  of animals of each sex shall  be used
at each concentration.

    (B) Females shall be nulliparous and nonpregnant.

    (iv) Numbers.  (A) At least 20 animals (10 females  and  10 males)
should be used for each test group.

     (B) If interim sacrifices are planned, the number of animals shall be
increased by the number of animals scheduled to be sacrificed before the
completion of the study.

     (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  shall be assigned a unique identification number.
Dead animals, their preserved organs and tissues, and microscopic slides
shall be identified by reference to the animal's unique number.

     (v) Husbandry. (A) Animals may be group-caged by sex, but the
number of animals per cage must not interfere with clear observation of
each animal. The biological properties of the test substance or toxic effects
(e.g., morbidity, excitability) may indicate a need for individual caging.
Animals must be housed individually in inhalation chambers during expo-
sure to aerosols.

     (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 30-70 percent.

     (D) Where lighting is artificial, the sequence should be 12 hours light/
12 hours dark.

     (E) Control and test animals should be fed from the  same batch and
lot. The feed should be analyzed to assure adequacy of nutritional require-
ments of the species tested  and  for impurities that might influence the
outcome  of the  test. For feeding,  conventional laboratory  diets may be
used with an unlimited supply of drinking water.

     (F) The study should not be initiated until animals have been allowed
a period of acclimatization/quarantine  to  environmental  conditions, nor
should animals from  outside sources be placed on test without an adequate
period of quarantine.  An acclimation period of at  least 5  days is rec-

     (2) Control and test substances,  (i) Whenever it is necessary to for-
mulate the test substance with a vehicle for aerosol generation, the vehicle
ideally should not elicit toxic effects  or substantially alter the chemical
or toxicological properties of the test substance.

     (ii) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability. Prior to the initiation
of the study, there should be a characterization of the test  substance, in-
cluding the  purity of  the test substance and, if technically feasible, the
name and quantities of unknown contaminants and impurities.

     (3) Control groups.  A  concurrent  control group  is required.  This
group shall be an untreated or sham-treated control group. Except for treat-
ment with the test substance, animals in the control group shall be handled
in a manner identical to the  test group  animals. Where a vehicle other
than water is used to generate a substance, a vehicle control group should
be used. If the toxic properties of the vehicle are not  known or cannot
be made available, both untreated and  vehicle control groups are required.

     (4) Satellite group. A satellite group of 20 animals (10 animals per
sex) may be treated with the high concentration level for 90 days  and
observed for reversibility, persistence, or delayed occurrence of toxic ef-
fects for a post-treatment period of appropriate length,  normally not  less
than 28 days. In addition, a control group of 20 animals (10 animals of
each sex) should be added to the satellite study.

     (5) Concentration  levels and concentration selection,  (i) In sub-
chronic toxicity tests, it is desirable to have a concentration-response rela-
tionship as well as a NOEL. Therefore, at least three concentration levels
plus a control and,  where  appropriate, a vehicle control (corresponding
to the concentration of vehicle at the highest exposure level) shall be used.
Concentrations should be spaced appropriately to produce test groups with
a range of toxic  effects. The  data should be  sufficient to produce a con-
centration-response curve.

     (ii) The highest concentration should result in toxic effects but not
produce an incidence of fatalities which would prevent a meaningful eval-

     (iii) The intermediate concentrations  should be spaced to produce a
gradation of toxic effects.

     (iv) The lowest concentration should produce no evidence of toxicity.

     (v) In the case of potentially  explosive  test  substances,  care should
be taken to avoid generating explosive  concentrations.

     (6) Administration  of the test  substance. Animals should be ex-
posed to the test substance for 6 h per  day on a 7-day per  week basis
for a period of at least 90 days. Based  primarily on practical consider-
ations, exposure for  6 h per day on a  5-day per week basis is acceptable.

     (7) Observation period. The animals should be observed for a period
of 90 days. Animals in the  satellite group (if used) scheduled for follow-
up observations  should be kept for at  least 28 days further without treat-
ment to assess reversibility.

     (8) Exposure specifications, (i)  The animals shall be tested in dy-
namic inhalation equipment designed to sustain a minimum airflow 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 the surrounding areas. It is not normally necessary to
measure chamber oxygen concentration if airflow is adequate.

    (ii) 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 to expose animals to an aerosol, 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 shall not exceed 5 percent of
the volume of the test chamber. It is recommended, but not required, that
nose-only or head-only exposure be used for aerosol studies  in  order to
minimize  oral exposures due to animals licking compound off their fur.
Heat stress should be minimized.

    (iii) The temperature at which the test is performed should be main-
tained at 22 ±2  °C. The relative humidity should be maintained  between
40-60 percent, but in certain instances (e.g., use of water vehicle) this
may not be practicable.

    (9) Physical measurements. Measurements or monitoring  shall  be
made of the following:

    (i) The rate of airflow  shall be monitored continuously but recorded
at least three times during the exposure.

    (ii) The actual concentrations  of the test substance shall be measured
in the animal's breathing zone. During the exposure period,  the actual con-
centrations of the test substance shall  be held as constant as practicable
and monitored continuously or intermittently depending on the method of
analysis.  Chamber concentration may  be measured using  gravimetric  or
analytical methods as  appropriate. If trial run measurements are reasonably
consistent (±10 percent for liquid  aerosol, gas, or vapor; ±20  percent for
dry aerosol), then two measurements should be sufficient. If measurements
are not consistent, three to four measurements should be taken. Whenever
the test substance is a formulation, or it is necessary to formulate the test
substance with a vehicle for aerosol generation,  the  analytical concentra-
tion must be reported for the total formulation, and not just for the active
ingredient (AI). If, for example, a  formulation contains 10 percent AI and
90 percent  inerts, a  chamber  analytical limit  concentration  of  2 mg/L
would consist of 0.2 mg/L of the  AI. It is not necessary to analyze inert
ingredients provided the mixture at the animal's breathing  zone is analo-
gous to the formulation; the grounds for this conclusion must be provided
in the study report. If there is some difficulty in measuring chamber analyt-
ical concentration due to precipitation, nonhomogeneous mixtures, volatile
components, or other factors, additional analyses of inert components may
be necessary.

     (iii) During the development of the generating system, particle size
analysis shall be performed to establish the stability of aerosol concentra-
tions with respect to particle size. The MMAD particle size range  should
be between 1-3 (im. The particle size  of hygroscopic materials  should
be small enough when dry to assure that the size of the swollen particle
will  still be within the 1-3 (im range. Measurements of aerodynamic par-
ticle size in the animal's breathing zone should be measured during a trial
run.  If MMAD values for each exposure level are within 10 percent of
each other, then two measurements during the exposures should be suffi-
cient. If pretest measurements are not within 10 percent of each other,
three to four measurements should be taken.

     (iv) Temperature and humidity shall be monitored continuously and
recorded at least three times during an exposure.

     (10) Feed and water during  exposure period. Feed shall be with-
held during exposure. Water may also be withheld during exposure.

     (11) Observation of animals, (i) During and following exposure, ob-
servations are made and recorded systematically; individual records  should
be maintained for each animal. It is not always possible to observe animals
during exposure in a whole-body chamber.

     (ii) Observations shall be made at least twice each day for morbidity
and  mortality. Appropriate actions should be taken to minimize loss of
animals to the study (e.g., Necropsy or refrigeration of those animals found
dead and isolation or sacrifice of weak or moribund animals).

     (iii) General clinical observations should be made at least once a day,
preferably at the same time  each day, taking into consideration the peak
period of anticipated effects after dosing. The clinical condition of the ani-
mal should be recorded.

     (iv) A careful clinical examination should be made at least once prior
to the initiation of treatment (to allow for within subject comparisons) and
once weekly during treatment in all animals. These observations  should
be made outside  the  home cage,  preferably in a standard arena,  and at
similar times on each occasion. Effort should be made to  ensure that vari-
ations in the observation  conditions are  minimal. Observations  should be
detailed and carefully recorded,  preferably using scoring  systems, explic-
itly defined by the testing laboratory. Signs noted should include, but not
be limited to,  changes in skin, fur, eyes, mucous membranes,  occurrence
of secretions  and excretions and  autonomic activity  (e.g., lacrimation,
piloerection, pupil size, unusual respiratory pattern). Changes in gait, pos-
ture  and response to handling as well as the presence of clonic or tonic
movements, stereotypies (e.g., excessive grooming, repetitive circling) or
bizarre behavior (e.g., self-mutilation, walking backwards) should be re-

     (v) Once,  near the end of the exposure period and  in any  case not
earlier than in  week 11, assessment of motor activity,  grip strength, and
sensory reactivity to stimuli of different types (e.g., visual, auditory, and
proprioceptive  stimuli) should be conducted. Further details of the proce-
dures that could be followed are described in the references listed under
paragraphs (h)(3),  (h)(4),  (h)(5), (h)(7), (h)(8), and (h)(ll) of this guide-

     (vi) Functional observations conducted towards the end of the study
may be omitted when  data  on  functional observations are available from
other studies and the daily  clinical observations did not reveal any func-
tional deficits.

     (vii) Exceptionally, functional observations may be omitted for groups
that otherwise reveal signs of toxicity to an extent that would significantly
interfere with functional test performance.

     (viii) Individual weights of animals shall be determined shortly before
the test substance is administered, and weekly thereafter.

     (ix) Food  consumption shall also be determined weekly if abnormal
body weight changes are observed.

     (x) Moribund animals should be removed and sacrificed when noticed
and the time of death should be recorded as precisely as possible.

     (xi) At termination, all  survivors in the treatment groups shall be sac-

     (12) Clinical pathology. Hematology and clinical chemistry examina-
tions  should be made  on all animals, including  controls, of each sex  in
each group. The hematology and clinical chemistry parameters should be
examined at terminal sacrifice  at the end of the  study. Overnight fasting
of the animals prior to blood  sampling is recommended. Overall, there
is  a need for  a flexible approach in the measures examined,  depending
on the observed or expected effects from a chemical, and  in the frequency
of measures, depending on  the duration of potential chemical  exposures.

     (i) Hematology.  The recommended parameters are  red blood cell
count, hemoglobin concentration,  hematocrit, mean corpuscular  volume,
mean  corpuscular  hemoglobin,  and mean  corpuscular hemoglobin con-
centration,  white blood cell count,  differential  leukocyte count, platelet
count, and a measure  of  clotting potential, such as prothrombin time  or
activated partial thromboplastin time.

     (ii) Clinical chemistry. (A) Parameters which  are  considered appro-
priate to all studies are electrolyte balance, carbohydrate metabolism, and
liver and kidney function.  The selection of specific tests will be influenced
by observations on the mode of action of the substance  and signs of clini-
cal toxicity.

     (B)  The recommended clinical chemistry determinations  are  potas-
sium, sodium,  glucose, total cholesterol, urea nitrogen, creatinine, total
protein  and albumin.  More  than 2 hepatic enzymes, (such  as alanine
aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol
dehydrogenase, or gamma glutamyl transpeptidase) should also be  meas-
ured. Measurements of addtional enzymes (of hepatic or other origin) and
bile acids, may also be useful.

     (C)  If a test chemical has an effect on the hematopoietic system,
reticulocyte counts and bone marrow cytology may be indicated.

     (D)  Other  determinations that should be carried out if the test chemi-
cal is known or suspected of affecting related measures include calcium,
phosphorus,  fasting   triglycerides,  hormones,   methemoglobin,   and

     (iii) Optionally, the following urinalysis determinations could be per-
formed during the last week of the study using timed urine volume collec-
tion: appearance, volume, osmolality or specific gravity, pH, protein, glu-
cose, and blood/blood cells.

     (13)  Ophthalmological  examination.  Ophthalmological  examina-
tions using an  ophthalmoscope or  an equivalent device should be made
on all  animals prior  to the administration of the test substance and  on
all high  dose  and control groups  at termination.  If changes in the eyes
are detected, all  animals  in the other dose groups  should  be  examined.

     (14) Gross necropsy, (i) All animals shall be subjected to a full gross
necropsy which includes examination of the external surface of the body,
all orifices and the cranial, thoracic, and abdominal cavities and their con-

     (ii) The liver, kidneys, adrenals, testes,  epididymides, ovaries, uterus,
thymus,  spleen, brain, and heart shall be trimmed and weighed wet, as
soon as possible after  dissection to avoid drying.

     (iii) The following organs and tissues, or representative samples there-
of,   shall 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 (where present).
     (B) Nervous system.
     (1} Brain (including sections of medulla/pons, cerebellum, and cere-
     (2) Pituitary.
     (3} Peripheral  nerve  (sciatic  or tibial,  preferably in close proximity
to the muscle).
     (4) Spinal cord (three levels: cervical, mid-thoracic, and lumbar).
     (5) Eyes (retina, optic nerve).
     (C) Glandular system.
     (1} Adrenals.
     (2) Parathyroids.
     (3} Thyroids.
     (D) Respiratory system.
     (1} Trachea.
     (2} Lung.
     (3} Pharynx.
     (4) Larynx.
     (5) Nose.
     (E) Cardiovascular/hematopoietic system.
     (1} Aorta.
     (2) Heart.
     (3} Bone marrow (and/or fresh aspirate).

     (4) Lymph nodes (preferably one node covering the route of adminis-
tration and another one distant from the route of administration).
     (5) Spleen.
     (6) Thymus.
     (F) Urogenital system.
     (7) Kidneys.
     (2) Urinary bladder.
     (3) Prostate.
     (4) Testes.
     (5) Epididymides.
     (6) Seminal vesicle(s).
     (7) Uterus.
     (8) Ovaries.
     (9) Female mammary gland.
     (G) Other.
     (7) Lacrimal gland.
     (2) Skin.
        All gross lesions and masses.
     (15) Histopathology. (i) The following histopathology shall be per-
     (A) Full histopathology on the respiratory tract and other organs and
tissues,  listed under paragraph (e)(15)(iii) of this guideline, of all animals
in the control and high exposure groups and all animals that died or were
killed during the study.
     (B) All gross lesions in all animals.
     (C) Target organs in all animals.
     (D) Lungs of all animals. Special attention to examination of the res-
piratory tract should  be made  for evidence  of infection  as this provides
a convenient assessment of the state of health of the animals.
     (E) When a satellite group is used, histopathology shall be performed
on tissues and organs identified as showing  effects in the treated groups.

     (ii) If excessive early deaths or other problems occur in the high expo-
sure group compromising the significance of the data, the next concentra-
tion should be examined for complete histopathology.

     (iii) An attempt should be made to correlate gross observations with
microscopic findings.

     (iv)  Tissues  and  organs  designated for  microscopic examination
should be fixed in 10  percent buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours prior
to trimming.

     (f) Data and reporting—(1) Treatment  of  results,  (i)  Data shall
be summarized in tabular form, showing  for each  test group the number
of animals at the start  of the test, the number of animals showing lesions,
the types of lesions, and  the percentage of animals displaying  each type
of lesion.

     (ii) When applicable, all observed results, quantitative and qualitative,
should be evaluated by an appropriate statistical method. Any generally
accepted statistical method may be used; the statistical methods including
significance criteria should be selected during the design of the study.

     (2) Evaluation  of study results. The findings of the subchronic inha-
lation toxicity study should be evaluated in conjunction with the findings
of preceding studies and considered in terms of the observed toxic effects
and the necropsy  and  histopathological findings. The evaluation will in-
clude the relationship between the concentration of the test substance and
duration of exposure, and the presence or absence, the incidence and sever-
ity, of abnormalities, including behavioral  and clinical abnormalities, gross
lesions, identified target organs, body weight changes, effects on mortality
and any  other general  or specific toxic effects. A properly conducted sub-
chronic test should provide a satisfactory estimation  of a no-effect level.
It also can indicate the need for an  additional longer-term study and pro-
vide information on the selection of concentrations.

     (3) Test report. In addition to reporting requirements specified under
EPA Good Laboratory Practice Standards, 40 CFR part 792,  subpart J and
40 CFR part 160,  and  the OECD principles of GLP (ISBN 92-64-12367-
9) the following specific information shall  be reported:

     (i) Test substance  characterization shall include:

     (A) Chemical identification.

     (B)  Lot or batch number.

     (C)  Physical properties.

     (D) Purity/impurities.


     (E) Identification and composition of any vehicle used.

     (ii) Test system information shall include:

     (A)  Species and strain of animals used  and rationale for  selection
if other than that recommended.

     (B) Age, sex, and body weight data.

     (C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods.

     (D) Identification of animal diet.

     (E) Acclimation period.

     (iii) Test procedure information shall include:

     (A) Method of randomization used.

     (B) Full description of experimental design and procedure.

     (C) Exposure regimen including levels, methods, and volume.

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

     (7) Description of exposure  apparatus including design, type, volume,
source of air, system  for generating  aerosols, method of conditioning air,
treatment of exhaust air and the method of housing the animals in a test

     (2) The equipment for measuring temperature, humidity, and particu-
late aerosol concentrations and size should be described.

     (E) Exposure data. These  shall be tabulated and presented with mean
values and a measure of variability  (e.g., standard deviation) and  should

     (7) Airflow rates through the inhalation equipment.

     (2) Temperature and humidity of air.

     (3) Actual (analytical or  gravimetric) concentration in  the breathing

     (4) Nominal concentration  (total amount of test substance fed into
the inhalation equipment divided by volume of air).

     (5) Particle size distribution, calculated mass median aerodynamic di-
ameter (MMAD) and geometric standard deviation (GSD).


     (6} Explanation as to why the desired chamber concentration  and/
or particle size could not be achieved (if applicable) and the efforts taken
to comply with this aspect of the guidelines.

     (iv) Test results. (A) Group animal data. Tabulation of toxic response
data by species, strain, sex, and exposure level for:

     (7) Number of animals exposed.

     (2) Number of animals showing signs of toxicity.

     (3) Number of animals dying.

     (B) Individual animal  data.  Data should be presented as summary
(group mean) as well as for individual animals.

     (7) Time of death during the  study or whether animals survived to

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

     (3) Body weight data.

     (4) Feed consumption data, when collected.

     (5) Results of ophthalmological examination.

     (6) Results of hematological tests performed. .

     (7) Results of clinical chemistry tests performed.

     (8) Results of urinalysis, if performed.

     (9) Results of observations made.

     (Iff} Necropsy findings, including  absolute and relative organ weight

     (11) Detailed description of all histopathological findings.

     (72) Statistical treatment of results, where appropriate.

     (g) Quality control.  A system shall be developed and maintained to
assure and document adequate performance of laboratory staff and equip-
ment. The study must be  conducted in compliance with GLP regulations.

     (h) References. The following  references should be consulted for ad-
ditional background information on this test guideline:

     (1) Cage, J.C. Experimental Inhalation Toxicology,  Methods in Toxi-
cology. Ed. G.E. Paget (Philadelphia: F.A. Davis Co.,  1970) pp. 258-277.


     (2) Casarett, L.J.  and Doull,  J. Chapter 9, Toxicology: The  Basic
Science of Poisons (New York: Macmillan Publishing Co. Inc., 1975).

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