HAZARD EVALUATION DIVISION

        STANDARD EVALUATION PROCEDURE

         INHALATION TOXICITY TESTING
                 Prepared by

  Stanley B.  Gross/ Ph.D., D.A.D.T., C.I.H,
              Toxicology Branch

                     and

          Frank J. Vocci, Consultant
Standard Evaluation Procedures Project Manager

     Orville E. Paynter, Ph.D., D.A.B.T.
          Hazard Evaluation Division
         Office of Pesticide Programs
United States Environmental Protection Agency^
         Office of Pesticide Programs
            Washington, DC  20460

-------
• 101
REPORT COCUMENTATION L. R PQRT NO. 2.
. PAGE P
3. RscJp u is1 OA No.

a flu. . $ubIiII•
Evaluation Bivision: Standard Eva1ua ion Procedure
• Th a ti n• Thxi .T sting
I. *.po O c
9ti.L t &
a
7. Aijthor(a)
Stanley B. Cross and Frank J. Vocci
a Ogu , R.oi.
540/0988—l01
I. P f iw k Or .nl ..t .n Nam . m d Add,...
U.S.. Environmental Protection Agertcy/OPP/HED/TS—769c
401 N Street SW
Washington, DC 20460
I P’V Thmi/W ., . U ’WI No.
.
mO .. .t(c N o.
‘

1.2. 3.oWn. Gvim&..ti.n N.m. aa A4dru . .
Same as t 9

13. Typ. R.povt Pio Cm. .o
1 4.
13. up01v ’iN.,y Not..
14. (Uaift O eur
The Standard Evaluation Procedure (SE PY?or Inhalation
Toxicity Testing discusses the requirements for inhalation
toxicity testing defined in the Agency Guidelines. It
discusses the different types of inhalation experiments that
are usua.lly carried out, the species usually used, exposure
and genelating systems, and the analyses of the exposure
atmospheres. The SEP explains the reasons for the different
requirements and how the requirements are usually met. There
is some discussion on dealing with various problems which
arise in these tests and a brief discussion on the bases for
rejecting an inhalation study.
17. Doaa , i y s a
3. Pu/Op.n.1i 7.,,,.
a cO$A11 fldd/Om.p
1$. Av01IiW tp 3IEU .f 4
.. NU I4
$i NoUI .. UAI
(? m.r? N 1
ol C

-------
STANDARD EVALUATION PROCEDURE
PREAMBLE
This Standard Evaluation Procedure (SEP) is
one of a set of guidance documents which explain
the procedures used to evaluate environmental arrd
human health effects data submitted to the Office
of Pesticide Programs (0??). The SEPs are designed
to ensure comprehensive and consistent treatment
of major scientific topics in these reviews and
to provide interpretive policy guidance where
appropriate. The SEPs will be used in conjunction
with the appropriate Pesticide Assessment Guide-
lines and other Agency Guidelines. While the
documents were developed to explain specifically
the principles of scientific evaluation within
OPP, they may also be used by other offices in
the Agency in the evaluation of studies and
scientific data. The SEPs will also serve as
valuable internal reference documents and will
inform the public and regulated comn unity of
important considerations in the evaluation of
test data for determining chemical hazards. I
believe the SEPs will improve both the quality
of science within EPA and, in conjunction with
the Pesticide Assessment Guidelines, will lead
to more effective use of both public and private
resources.
Anne L. Barton, Acting Director
Hazard Evaluation Division

-------
TABLE OF CONTENTS
I • PREFACE . . . . . . • . . . • . . . .
II. INTRODUCTION . . . . . . . . . . . . . • •
A. Single Short—term Exposures . . .
B. Repeat Inhalation Exposure Studies
C. Inhalation Exposure Systems . . .
III. INHALATION CHAMBER EXPOSURES • . . . .
A. Test Substances . . . . . . . . .
B. Test Species . . . . . . • . • • .
C. Test Chambers . . . . . . . . . .
1. Chamber Designs . . . . . . .
2. Animal Loading .
3. Air Flow Rates . . . . . . . •
4. Chamber Temperatures . . . . .
5. Chamber Humidity . . . . . . . .
6. ChamberOxygen. . . . . . . .
7. Chamber Equilibration . . . . .
D. Generation Systems . . . . . . . • . . •.
1. Generation of Gaseous Atmospheres .
2. Generation of Vapors . . . . . . . .
3. Generation of Sprays
4. Generation of Dusts . • . . . . . .
C. Chamber Concentrations . . . . . . . . .
1. Concentration Measurement Methods
2. Indirect Methods of Analysis . . .
3. Frequency of Measurement . . . . .
F. Particle Size Determinations . . . . . .
1. Aerodynamic Diameters . . . . . . •
2. Measurement of Aerodynamic Diameters • .
IV. ALTERNATIVE METHODS FOR INHALATION EXPOSURES
Page
• . . 1
• . . 2
• . . 2
• . . 2
• . • 3
. • . 4
• . . 4
• S 5
. . . 5
• . • 5
• S 5 7
• S 7
• S 5 7
• . . 8
• • . 8
• . • 9
.9
10
10
10
11
12
• 12
• 13
• 13
13
14
14
17

-------
TABLE OF CONTENTS (Cont’d)
Page
• • . . . • . 18
• . • . . . . 18
• . S • S S • 18
• S • • S • • 19
• S S • S 5 5 20
V. ANIMAL OBSERVATION . . . • • • .
A. Routine
B. Lung Function Studies • • . .
VI. ACCEPTABILITY OF INHALATION STUDIES
REFERENCES • • . . . • . • . • . • , • .
i;/

-------
TABLE S
Tables Page
I Overview of Acute Inhalation Toxicity
Testing . . . . . . . . . . . . . • • • • • T1
2 Overview of 90—Day Inhalation Toxicity
Testing . . . . . . . . . . . . . . . . . . . . T—4
3 Some Physiological Indices of Man and
Animals . . . . . , . . . . . . . . . . . . . . T—7
4 Output Characteristics of Some Compressed
Air Nebulizers . . . . . . . . . . . . . . . . T—8
5 Sample Particle Size Data . . . . . . . . . . . . T—9
6 Routine Clinical Laboratory Tests . . . . . . . . T—lO
7 Complete Gross Necropsy and Histopathological
Examinations . . . . . . . . . . . . . . . . . T—ll

-------
FIGURES
Figures Page
1 Components of Simplified Exposure System . . . . F—i
2 Schematic Diagram of the Rochester Exposure
Chamber . . . . . . . . . . . . . . . . . . . F—2
3 Schematic Diagram of the New York University
ExposureChamber . . •....... •... F—3
4 Equilibration of Exposure Chambers . . . . . . . F—4
5 Diagram of the DeVilbiss Nebulizer . . . . . . F—5
6 Aerodynamic Size (Diameter) vs. Deposition in
the Lung . . . . . . . . . . . . . . . . . . . F—6
7 Schematic View of Cascade Impactor . . . . . . . F—7
8 Example Particle Size Graph . . . . . . . . . . F—8
v

-------
I. PREFACE
Pesticides are often formulated and applied as gases,
vapors, sprays, or dusts and therefore present possible exposures
to humans by the inhalation route. Requirements for pesticide
registration (40 CFR Part 158) identify the types of pesticide
uses that require inhalation toxicity testing involving animals.
Guidelines for such testing have been provided by the Agency’s
Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation;
Human and Domestic Animals.
This Standard Evaluation Procedure (SEP) focuses on the
inhalation testing systems that are required for animal inhalation
tests. These systems offer a number of technical difficulties
beyond testing by the oral and dermal routes. Chamber operations
and atmosphere generation equipment are expensive and complicated.
Atmosphere characterization is less well understood and is often
difficult to assess. The difficulties may be further compounded
because today’s requirements are more stringent than those estab-
lished in previous years. There are cited in the literature a
larger variety of techniques and equipment which are useful for
research in inhalation toxicology but are not typical of the
techniques used in commercial inhalation research facilities.
This discussion will therefore focus on the inhalation
testing methods and equipment that are typical of those used to
meet the requirements for inhalation testing for pesticide regis-
tration. It is assumed that the reader has a general background
in pulmonary anatomy and physiology as well as the rudiments of
inhalation testing systems. A number of references cited can
provide additional background for those who need it.
1.

-------
II. INTRODUCTION
Subpart F provides two guidelines which are specifically
for inhalation toxicity testing: the acute study (S81—3) and the
subchronic inhalation study (582—4). Previous discussion of the
Guidelines for inhalation testing have been published by the
principal author (Gross, 1981 and 1987). Tables 1 and 2 provide
overview of the Guideline requirements for these two types of
studies. The acute inhalation study involves single short—term
exposure and the subchronic study involves repeat exposures of
intermediate duration.
A. Single Short—term Exposures
The acute inhalation toxicity test is required by 40
CFR Part 158 to support the registration of a pesticide product
if it is a gas, a solid, or a liquid with a significant vapor
hazard, contains particles with aerodynamic diameters of 15
micrometers or less, or when applied it will produce particles
of 15 micrometers or less. The acute inhalation toxicity test
provides information on health hazards likely to arise from
single exposures. It is used to determine the median lethal
concentration (LC 50 ), its statistical limits and slope using a
single exposure, usually 4 hours, and a 14—day postexposure
period. It is also the initial step in establishing a dosage
regimen for subchronic and other studies and may provide informa-
tion on the mode of toxic action of the agent.
The acute inhalation study represents the type of
exposure technique that could be used in other types of studies
in which single inhalation exposures are used——such as the acute
delayed neurotoxicity study (S81—7) or metabolism studies (585—1).
B. Repeat Inhalation Exposure Studies
•Repeat• inhalation exposure studies refers to
exposures repeated daily usually from 2 weeks to the animal’s
lifetime (2 years for rodents). The exposure methods used in
repeat studies are similar. The subchronic inhalation toxicity
study (580—4) describes the testing requirements aimed at 90—day
inhalation exposures. The exposure techniques here describe
chamber exposures of 6 hours/day, 5 days/week. Similar inhalation
exposure methods are used for chronic exposures (583—1), oncogeni—
city studies (583—2), teratology studies (S83—3), and reproductive
and fertility effect studies (580—4) in which the exposures will
vary with the respective Guideline and its intended purpose.
The present discussion focuses on the inhalation
exposure methods and analyses in the context of the requirements
for the Acute and Subchronic Inhalation Toxicity Testin,g Guidelines.
It will emphasize those issues which relate to inhalation testing
2

-------
and inhalation testing systems and recommends that the reader
consult specific SEPs (such as those for chronic studies, oncoge—
nicity studies, and teratology) when such studies also involve
inhalation route testing.
C. Inhalation Exposure Systems
Whole—body inhalation exposure chambers are g nera1ly
used for studies submitted to the Agency. Subpart F Guidelines
are written with such chambers in mind. Alternative methods of
exposures including head oniy, nose/mouth only, lungs only, and
partial lung systems are discussed in some detail by Phalen
(1984). Whole—body chambers come in a wide variety of sizes,
materials, and designs (from small cylinders to walk—in rooms).
Whole—body exposure chambers have advantages in that they involve
a minimum of constraint for the animals, allow for controlled
environments and are suitable for chronic studies. On the other
hand, they are expensive, inefficient, provide multiple routes of
exposure, and poor contact between animals and investigators.
The other exposure methods provide more control of the exposures
to the lungs, control of the contaminants, and provide efficient
utilization of test materials. However, there may be stress to
the animals, which may cause aberrant breathing patterns, leakage
of the exposure systems which causes problems, so that they have
not been suitable for long—term exposures.
The Guidelines focus on whole—body inhalation chambers;
however, the tester has the option of using other systems when
justification can be established.
3

-------
III. I *IALATION CHAMBER EXPOSURES
The objectives of the chamber exposure systems are to
provide uniform, controlled exposures that would not produce
harmful effects without the test agent. In acute exposure
studies, the different test levels do not have to be run
concurrently. In the repeat studies, the control and exposure
chambers should be of the same make and design and all test
groups shoula be run concurrently under the same environmental
conditions.
A. Test Substances
The test substance will vary depending upon form of the
pesticide, i.e., the technical grade, manufacturing—use product,
end-use product or an end—use dilution. The presence of test
materials or conversion products in gaseous or aerosol form is of
concern as long as an inhalation hazard exists, whether by direct
application or reentry. If the purpose of the study is to evaluate
the inhalation hazards associated with reentry into a treated
area, the test substance may need to be made up of conversion
products representative of those to which workers may be exposed.
The physical and chemical form of the test material
should be similar to that to which man or animals will be exposed.
Available inhalation studies show that the absorption of gases or
vapors is driven by the laws of diffusion and thermodynamic
activity. The concentration gradient and partition coefficient
(air/tissue) play a significant role in the amount of material
inhaled and absorbed. Soluble gases tend to be absorbed in the
upper respiratory tract and as solubility decreases, the potential
for penetration into the deeper portions of the lung increases.
For different reasons, a similar situation to gases and
vapors arises from the inhalation of aerosols. Both inertial and
diffusional forces operate primarily (sometimes simultaneously)
for the deposition of particles in the respiratory tract. Moreover,
regional deposition in different species is a function of particle
size (evidence for man i5 better developed than for other animal
species), so that the smaller the particle the greater the proba-
bility of reaching the deeper (alveolar) region. Regional deposi-
tion for different animal species is a function of particle size
and animal size, so that a product made up of large particles
will require reduction in particle size for smaller animals.
In some cases, a vehicle (such as water or an organic
solvent) may be necessary in order to produce an aerosol of the
test material. Such agents should be relatively nontoxic and
should not significantly affect the biological properties of the
test materials. Some vehicles are toxic enough to enhaflce the
toxicity of a test substance so as to give the impression that
the test material is more toxic than it is. This is an important
4

-------
consideration in the case of acute Inhalation tests which do not
require vehicle controls.
B. Test pecies
It is desirable to select the species which are
anatomically and physiologically similar to humans. Table 3
provides a comparison of a number of parameters between ?nan and
animals. Since no single animal species is anatomically similar
to man in all respects, researchers have used mice, rats, monkeys,
rabbits, hamsters, guinea pigs, Cats, and dogs for routine studies.
Young adult male and female laboratory rats are usually
recommended for inhalation studies discussed in the Guidelines.
Rats are readily available, inexpensive and a considerable data
base has been established for the different varieties used. If
another mammalian species other than the rat is used, the tester
should provide the justification for its selection; the primary
ustlfication would be metabolic similarity.
C. Test Chambers
1. Chamber Designs
Whole—body exposure chambers are static or dynamic
in design. A static exposure chamber consists of a closed chamber
into which a predetermined amount of the test agent is introduced
in the chamber to produce a predetermined concentration. The
test animals remain in this closed system for the duration of
test without any replacement of air. The lack of replacement air
limits the duration of the exposure and allows for an increase of
carbon dioxide levels, increased temperature from the buildup of
animal body heat and increased humidity. There is a loss of test
substance with time to the animals and chamber surfaces. Chamber
concentrations are usually not determined because sampling affects
the concentration of the material inside the chamber. However,
an integrated exposure level is sometimes determined by taking
small chamber sample5 periodically, which provide a decay curve.
Static chambers are used primarily as hazard tests
for gases and vapors. They are not used in Guideline studies but
may be useful for determining range—finding levels prior to
dynamic chamber studies.
A dynamic exposure chamber consists of a chamber
that has a continuous flow through of air and test agent at a
constant rate and in a predetermined concentration. Figure 1
represents a dedicated chamber exposure system which reflects the
types of equipment and processes necessary to support and monitor
the whole—body inhalation chamber. The air provided directly to
the chamber and air used during the generation of the test systems
generally goes through a treatment system whereby it is filtered,
5

-------
scrubbed if necessary, cooled and/or heated and otherwise condi-
tioned. This air flow is usually carefully metered,, often with
alarm systems which indicate problems of temperature and/or flow
into the chamber. Waste air from the chambers is also conditioned
by filters with charcoal beds, scrubbers or combustion chambers
which remove reactive chemicals before the air is vented to the
outside. The air in the chamber is controlled by a push—pull
system in which the incoming air pressure is lover than -the
exhaust pressure preventing the loss of test material from the
chamber into surrounding rooms.
Figure 1 depicts the complexity of systems dedicated
to inhalation testing. Smaller, less complicated systems, which
fit inside of hoods, are often used for short—term acute inhala-
tion testing involving high concentrations and smaller numbers
of animals. In this case, elaborate air conditioning systems are
not used but comparable chamber temperatures and humidity are to
be maintained. Chamber pressures in these systems are often
positive to surrounding pressures in order to prevent dilution of
the concentrations used inside of the chamber.
The chambers are usually constructed of stainless
steel and glass and/or clear plastic. Materials of porous con-
struction should be avoided as they absorb test materials which
are difficult to remove and may affect subsequent studies. The
chambers should be designed so that the animals are visible
during the exposure periods and can be readily removed for obser-
vations and sanitary maintenance. Ports are provided for sampling
the Internal environment of the chamber.
Two types of chambers, the Rochester (Figure 2) and
the New York University (Figure 3), are frequently used and are
commercially available. The Rochester chamber is a hexagonal—
shaped chamber with conical additions to the top and bottom. The
New York University chamber is based on a cubicular shape with
pyramidal additions. The Rochester and New York University cham-
bers are designed to help ensure a uniform distribution of test
atmospheres within the chambers. There are a variety of other
chambers that have been developed primarily to ensure uniform
exposure to the animals (see Leong 1982; Phalen 1984; and Drew
1979 for a discussion of several of these). The study report
should provide sufficient detail concerning the chambers used
so that the reviewer has a good understanding of the suitability
of the chamber for inhalation studies. If the investigating
laboratory uses a chamber of unfamiliar design and construction,
the laboratory needs to provide information to demonstrate its
usefulness, typically involving sampling from different locations
throughout the chamber and demonstrating uniform levels with time.
It is a good idea to check the uniformity of the atmosphere in
chambers for each new agent generation process because iifferent
agents can cause unexpected variability inside of routinely used
chambers (such as the Rochester or New York University chambers).
6

-------
Although somewhat dated, the novice to inhalation
testing systems will find useful the detailed discussion on
inhalation testing systems provided by Fraser et al. (1957).
2. Animal Load j
The animals are usually placed in the chambers in
stainless steel wire mesh cages, one animal per cage. Current
Guidelines call for only one species of animal in the chamber.
Experience has shown that animals should occupy 5 percent or less
of the volume of the chamber. This is calculated assuming each
gram of animal occupies I cc of volume within the chamber. With
chamber flow rates of 10 to 15 changes per hour, this ratio helps
to assure that the chamber temperature will not climb during
exposures, that oxygen levels will be adequate and the waste
products (such as carbon dioxide, water, and ammonia) will not
accumulate to uncomfortable levels for the animals or interfere
with the distribution of the test materials inside of the chamber.
During repeat daily exposures, animal cage locations
should be randomly rotated on a regular basis (at least weekly)
usually during daily maintenance procedures. This helps to
ensure that the animals’ exposure conditions will be more uniform.
Animal wastes are usually removed in the morning before test
atmospheres are turned on.
3. Air Flow Rates
Air flow into the chamber and through the generating
system must be calibrated during the development phases of the
study and monitored during the experimental stages. The Guidelines
for the acute inhalation study call for chamber air flow rates of
at least 10 changes per hour and for the repeat exposures, 12
changes per hour. Air flows of 10 to 60 chamber volumes per hour
can be found in the literature; however, high flow rates consume
large quantities of test material and require more resources in
recovering the test material on the waste discharge side of the
chamber. The measurement of flow in the acute study should be at
least every 30 minutes during the 4—hour exposure period and at
least once every hour for repeat exposures of a 6—hour daily
exposure. Flow rates are usually monitored using such devices as
rotameters or orifice meters during the exposure runs; however,
such devices may need to be calibrated using primary air flow
standards such as wet test meters, dry gas meters, standardized
rotameters or bubble meters using appropriate calibration methods.
4. Chamber Temperatures
The Guidelines indicate the chamber temperature
should be maintained at 22 °C (+ 2 °C). This is one ofseveral
temperatures ranges which are suitable for rats. Animal ventila-
tion rates and depth varies with different temperatures. The
7

-------
important concern here is that the temperature be maintained
under suitable control during the conduct of the study. When
other species other than the rat are used, the temperature in the
chamber may need to be changed. This should be done according to
the latest specifications of the National Academy of Sciences
(1972).
The Guidelines recommend temperature recording
every 30 minutes for acute studies and every 60 minutes for
repeat studies. The chamber temperature may be measured by a
simple thermometer and the measurement recorded manually. Most
often, the temperature is recorded remotely using a thermistor
sensor and an automatic recording device. Often the thermistor
is equipped with an alarm system which goes off if the temperature
goes beyond preset extremes of cold or heat. Precise calibration
of temperature sensing equipment is not usually required but
should be checked at least once daily with a laboratory thermometer
to assure the system is not malfunctioning.
5. Chamber Humidity
The Guidelines call for chamber humidity to be
controlled within a range of 40 to 60 percent. Low humidity can
adversely affect normal pulmonary function, especially clearance
of particulate material up the niucociliary escalator. A con-
trolled humidity range is suggested also because differences in
humidity can cause differences in the chemical and physical
characteristics of the test materials, especially the aerodynamic
behavior of particles. High humidity approaching 100 percent is
inevitable when the test substance is suspended in water.
Chamber humidity is most often controlled by
controlling the humidity of the chamber make—up air before it
gets to the chamber. Wet/dry bulb thermometers are frequently
used to control facility air before the air is later introduced
into the chamber. Various devices have been used for measuring
humidity inside of chambers, including recently available elec-
tronic devices. Devices for measuring humidity should be checked
against a primary standard (8uCh as a sling psychrometer) and
must be routinely cleaned and maintained in order for it to work
properly. Such instruments may not be suitably used inside of a
chamber because they may get covered with excessive quantities of
test material.
6. Chamber Oxygen
The Guidelines call for chamber oxygen concentrations
of at least 19 percent. Many laboratories do not measure chamber
oxygen directly. Adequate oxygen is generally provided when the
flow rates into the chamber are maintained. It is possible,
however, to push the chamber oxygen levels below 19 percent when
using low molecular weight gases at high concentrations (at the
8

-------
limit test level of 5 mg/L) and theoretically if the generated
test material consumes oxygen when it is introduced into the
chamber intake air.
Chamber oxygen can be measured using gasometric
techniques (the Orsatt), volumetric techniques (Van Slyke apparatus)
or more recent ion specific electrodes. These methods require
proper calibrations with standard gases.
7. Chamber Equilibration
In normal use situations, it takes a certain amount
of time for chambers to reach the desired concentration levels.
The concentration of a contaminant will rise and fall according
to an exponential function:
t K (a/b)
where t, is the time required to achieve x% of the desired
concentration, K is a constant for x% of the desired concentra-
tion, a is the volume of the chamber, and b is the volume of air
passing through the chamber per minute. The time to attain a
given concentration does not depend on the starting concentrations.
Figure 4 depicts the relationship of concentration within the
chamber with time. The time it takes to reach t 99 , K has a value
of 4.6 (99% of the desired concentration). With 10 to 12 air
changes per hour, steady state is reached in 20 to 25 minutes.
In previous times when 1—hour acute exposure studies were used,
many laboratories stopped the exposure 60 minutes after placing
the animals in the chambers. Therefore, one—third of the exposure
was below the expected concentrations at these stated air changes.
The simplest adjustment for equilibration times
is to extend the exposure time to allow for equilibrium, that
is, adding the equilibration time to the required exposure time
as seen in Figure 4. Alternative approaches are available but
usually require the availability of expensive monitoring equip-
ment. Because the t 99 value depends on chamber volume and flow
rate, it is possible to reduce the time to equilibrium by increasing
the flow for both the test agent and the total flow above the
required flow rate and reduce these when t 99 levels are achieved.
D. Generation Systems
The generation system refers to the equipment used to
introduce the test material into the chamber and will vary
depending on the physical characteristics of the test material.
A wide variety of methods are described in the literature many
of which are very specialized. All generating systems must go
through a development phase which provides experience w ith the
control of generation variables and the confirmation ofthe
reliability of the system itself. Calibration of the metering
9

-------
devices, maintenance and other precautions of importance will
be specific to the equipment being used. It is beyond the scope
of this discussion to go into much detail on the characteristics
of different generating systems. For additional background infor—
ination, the reader should consult such monographs as Fraser et al.
1957, Davies 1961, Mercer 1973, NIOSH 1973, and Leong 1981.
Although the equipment and processes for generating test atmospheres
can be technically complex, the •proof” of the reliabilfty of any
generation system 18 the production of well—controlled atmospheres
within the chamber as indicated by the concentration measurements
and, in the case of aerosols, the measurements of particle sizes.
1. Generation of Gaseous Atmospheres
These are usually simple to generate. If the
gas is available in a pressurized cylinder, it can be metered
through a flowmeter into the airstream entering the chamber where
it is then well mixed with the chamber make—up air before reaching
the animal breathing zone. The construction materials used in
the generation system should preclude problems of adsorption or
absorption. If the concentration of the gas to be used is quite
low, the gases may need to be diluted at atmospheric pressures
and metered into the airstream using a precision pump, such as a
syringe, piston pump, or peristaltic pump or premixed with a carrier
gas (such as air or nitrogen).
2. Generation of Vapors
Generation of vapors from liquid or solids often
involves simple devices. Vapors from solids can be generated by
passing a carrier gas (air) over or through a granular bed of
the material. Vapors from highly volatile liquids may be generated
simply by entraining dry air or nitrogen through a bubbler or by
passing air over a needle attached to a syringe activated by a
syringe pump. Often heat is applied to the liquid to increase
the vaporization rates. The amount of heat used in vaporization
may be crucial for organic materials which are heat sensitive. A
super—saturated vapor stream which moves into cooler parts of the
system may produce aerosols or condense on the surface of the
transfer lines and the chamber itself.
3. Generation of Sprays
Sprays or liquid aerosols consist of particles of
liquids suspended in air. Many pesticides are actually suspensions
of solids in liquids that are applied as sprays and therefore
need to be tested as sprays. The fact that the liquid is a
suspension of a solid and in cases of viscous liquids as the
suspending agents, generation of test sprays may be difficult to
impossible to achieve.
10

-------
Air—driven nebulizers or atomizers are often used
to produce polydisperse aerosols, Ultrasonic nebulizers are also
available for this purpose. Polydisperse aerosols have particles
with a range of particle sizes desirable for the purposes of
pesticide testing. In Contrast to the polydisperse aerosols are
generators which produce monodisperse aerosols in which the
particle size range is restricted. Monodisperse aerosol generators
include such devices as Sinclair—LaMer type generators,spinning
disk and top generators, vibrating reed, jet or orifice generators
and the like (Phalen 1984), and find their usefulness in research
to evaluate where in the lung particles are deposited.
Figure 5 shows the design of one of the commonly
used nebulizers and Table 4 lists some of the characteristics
for a number of the commonly used nebulizers. The basic nebulizer
uses compressed air, which is forced through a small orifice at
high velocity. The expansion of the gas leaving the orifice
creates a vacuum, which aspirates liquid up a small—diameter
tube and into the airstream. The airstream hitting the fine
column of liquid disperses droplets of the variable size and
carries them away. Impaction on the walls or a baffle inserted
in the airstrearn removes the larger droplets so that only the
smaller droplets are carried away.
It should be noted that the particle sizes
generated by the nebulizers shown in Table 4 are too large for
routine rodent inhalation testing if these are the sizes of the
liquid particles that reach the animals inside of the exposure
chambers. Median diameters of 1.0 urn are desirable. Particle
sizes delivered to the chamber may be reduced by evaporation
or increased by hygroscopicity. The latter may be removed
from the intake airstrearn by the use of a separating device such
as a cyclone separator of proper design.
4. Generation of Dusts
Dusts are aerosols of solid particles in air.
They are usually the most difficult materials to generate and
frequently offer problems that require unique solutions. A few
of the problems are difficulty in grinding the material into
small enough particles or the particles may agglomerate due to
electrostatic charges or hygroscopic nature. Frazer et al.
(1957), Mercer (1973), Leong (1981), and others provide discussions
on the use of a number of different systems that have been reported
in the literature.
A device frequently used for the generation of
dusts of pesticides is the Wright Dust Feeder. In this device,
a column of compacted powder is pushed against a rotating scraper
blade. The blade shaves off small quantities of the powder, and
this is entrained within a stream of air moving past the scraper.
The device is capable of producing high concentrations of dust
11

-------
particles. One problem occasionally encountered is variable
output related to nonuniform packing of the dust column, both
within a given column and between columns.
E. Chamber Concentrations
The Guidelines require the determination of actual
concentrations of the test substance in the atmosphere t the
breathing zone of the animals. In the past, the reporting of
nominal concentrationsD only was acceptable. ‘Nominal concen-
trations are calculated by determining how much of the test
substance was introduced into the chamber through the generating
system divided by the amount of air that was introduced into the
chamber. The actualN concentrations are concentrations based on
samples taken from the breathing zone of the animals and analyzed
by chemical or physical means. Most often the analytical concen-
tration is some fraction of the nominal concentration because of
a variety of possible reasons; the generating system may not be
able to generate an appropriate aerosol, much of the aerosol
generated may get deposited within the generation lines .eading
into the chamber, the flow and distribution through the chamber
may not be even, or the nature of the particles may be such that
they agglomerate into large particles that fail to remain airborne.
Gases and vapors may also be diminished from unominaiw due to
adsorptive and absorptive losses. Therefore, nominal concentra-
tions cannot be used to represent the concentrations to which the
animals have been exposed. However, it is a good idea to report
both nominal and actual concentrations because marked differences
between the two frequently reflect on the difficulty in keeping
an aerosol airborne.
1. Concentration Measurements Methods
The methods and equipment used for analytical
determinations of test atmospheres can vary considerably. Methods
used for gases and vapors will usually differ from those of
sprays and dusts. They will also vary for different chemical
species. Generally samples are drawn through sampling limes from
within the chambers, will be collected on solid media such as
charcoal or in a solution such as a solvent which will be trans-
ferred to a chemical laboratory for analysis. Many factors must
be considered when evaluating such methods, such as sampling effi-
ciency, chemical stability on the collecting media, efficiency of
recovering the test material from the collection media, and the
applicability of the analytical methods for the test materials.
For the latter, information on the sensitivity, precision and
accuracy should be discussed in the protocol. Details on these
issues are beyond the scope of this discussion, however, these
analytical methods should be examined to see if questions
concerning the methodology should be referred to one of the
Agency’s chemists.
12

-------
2. Indirect Methods of Analysis
A number of methods have been used which are not
specific for the test agent and are referred to as indirect
methods of analysis. They are useful because of the ease with
which they are carried out or because they provide the advantages
of real—time measurement of chamber concentration. An çxample of
the first type is that of collecting particles on filter media
and weighing the collected material when the aerosol particles
are not volatile. The concentrations are calculated from the
weight of the material collected divided by the volume of air
that passed through the filter. The collected material may
include substances other than the test substances. Therefore,
to determine the proportion of the dust represented by the test
agent, the sample should be analyzed chemically for the amount
of the test substance contained in the collected dust.
Real—time concentration measurements can be
achieved by drawing chamber air through an instrument which
measures the test agent by physical means such as the absorption
of infrared or ultraviolet light, conductivity and the like.
These physical measurements used for detection however, are not
specific.
For example, an infrared spectrophotometer can be
used to provide minute—to—minute estimates of the concentrations
inside the chamber but is not necessarily specific for the test
material itself. The wavelength chosen for the test material may
also be sensitive to carbon dioxide and moisture in the chamber.
When indirect measures of chamber concentration are
used, the sensitivity and precision of the methods need to be
compared to analytically specific methods.
3. Frequency of Measurement
Determinations should be made often enough to
adequately characterize the atmospheres to which the animals are
exposed. The degree of concentration variability and therefore
the frequency of determinations should be established during the
development of the generating system. The Guidelines, however,
specity the minimum frequency of these determinations. In the
case of the acute inhalation studies this is to be done at a
minimum of twice, one after initial chamber equilibration and one
late in the exposure period while the chamber is still at equili-
brium (see Figure 4). In the case of repeat exposures, the
analyses are to be carried out at least once per day.
F. Particle Size Determinations
When test agents are generated as aerosols, particle
size determinations are required to show that the test agent is
13

-------
small enough to be inhaled by the animal. This is especially
important when the exposures seemed to produce no adverse effects
on the exposed animals. It is possible to generate chamber aerosols
of high concentrations with particles that are so large that very
few will gain access to the pulmonary system during the test proce-
dures. It is important that the aerosol particle sizes are small
enough that the inhaled particles can reach the deeper portions
of the lung, that is the alveoli.
1. Aerodynamic Diameters
A rneas .irement of particle size that is most reliably
related to the ability of a particle to be inhaled and distributed
throughout the respiratory system is the aerodynamic particle
size. This is a concept which allows the standardized comparison
of particles of different shapes and densities. The aerodynamic
diameter is that diameter of a particle of unit density that has
the same terminal settling velocity as the particle in question.
Figure 6 shows the distribution of particles in the respiratory
system of man divided into three major areas——the nasopharyngeal,
the tracheobronchial and the alveolar regions. Large particle
(10 to 100 urn) sizes are deposited primarily in the nasopharyngeal
region usually by the process of impaction. Particles of 1.0 to
10 urn are deposited primarily in the tracheobronchial region,
usually by gravimetric sedimentation while small particles (1.0
urn and less) are deposited in the deep lung primarily by diffusion.
The deep lung provides a large surface (estimated at 10 sq meters)
for direct absorption of small particles while a substantial part
of the particles deposited in the upper portions of the lung can
be carried up the mucociliary escalator and swallowed. Where in
the respiratory tract and how much of the inhaled material is
deposited can substantially alter the toxicity of a test agent.
2. Measurement of Aerodynamic Diameters
There are many different ways of measuring particle
size diameters; however, only those methods that measure aerodynamic
diameters are to be used for inhalation toxicity testing. It is
important to note that particle size measurements based on optical
or electron microscope determinations or light scattering methods
do not provide measurements of aerodynamic diameters.
The cascade impactor shown in Figure 7 is often
used to determine aerodynamic diameters. This device uses the
principle of differential impaction to separate particles into
different size ranges. Standardized air flow through the impactor
carries the air through a series of stages with each successive
stage containing openings that are smaller than the previous
stage. Beneath each stage is a collection surface. As the aerosol
moves through the first stage openings, the airstream i s forced
to bend. The largest particles with sufficient momentum escape
the airstream and impact on the collection surface. The smaller
14

-------
particles with less momentum follow the airstream and move through
the openings in the next stage where another size cut is removed
and so on until the smallest particles are collected on a backup
filter. The particles deposited on each stage are of similar
aerodynamic size.
art
The particles collected on each of the impactor
stages are weighed and the weights are plotted on 1og—n rmal
graphs to determine the geometric mean and geometric standard
deviation of the particle size distributions. Table 5, Figure 8
shows some sample data that are obtained from the use of the
cascade impactor. The weight of the particles from each of the
stages from the impactor are added up and the cumulative percent-
age is calculated as shown in Table 5. The ECD represents the
average particle size determination for each of the stages. The
cumulative percentage data have been plotted on log—probability
coordinates (Figure B) which gave a aerodynamic diameter of 0.97
urn and a geometric standard deviation which was calculated by
dividing the diameter at 84 percent by the diameter at the 50
percent sizes to get a geometric standard deviation of 1.37. It
is possible to determine the geometric means and standard devia-
tions by calculating the logarithm of the stage size versus the
percent distribution of the particles.
As defined in the inhalation testing Guidelines
(and as shown by the example above), the ‘geometric mean diameter’
or ‘median diameter’ is that diameter that divides the particles
of an aerosol in half based on weight. Fifty percent of the
particles by weight will be smaller than the median diameter and
50 percent of the particles will be larger. The median diameter
usually used in rodent studies is around 1.0 micrometer with
geometric standard deviations approximately 2.5 or less. However,
the relationships of particle size and deposition in laboratory
rodents have not been adequately characterized. It would seem
appropriate that at least 25 percent of the particle distribution
used in these studies should be in the submicron range for acute
and repeat exposure studies.
When studies are carried out using large particle
distributions (median diameters greater than 3.0 urn), judgment
is necessary in determining whether the study should be repeated
using a smaller particle size range. If the chemical proved
quite toxic (Toxicity Category I, 40 CFR 162.10*), no further
acute testing is necessary as the chemical will already require
the strictest labeling. If the test results show minimal toxicity
by the inhalation route while showing significant toxicity via
other routes, then the acute inhalation testing should be
repeated using smaller particle sizes.
*Sectlon 162.10 of Title 40 of the Code of Federal Regulations.
15

-------
Adequate data on appropriate particle size selection
for inhalation studies is not available. Some reports in the
literature (such as that of Snipes et al. 1983) suggest particle
sizes of up to 3 urn can be used in rodents; however, these studies
report optical diameters rather than aerodynamic diarnete and
also report very small deposition rates (2.1%) of such p ides
in the deep lung.
The particle sizes recommended here are a matter
of judgment based on reviews of the different pesticide inhala-
tion studies from a number of laboratories over the years
and also from personal discussions with a number of prominent
inhalation toxicologists (including Drs. Robert Phalen of
University of California and Gerald Kennedy . f E.I. du Pont
de Nemours & Company).
16

-------
IV. ALTERNATIVE METHODS FOR INHALATION EXPOSURES
The methods described in detail here apply to whole—body
inhalation exposures because these are the systems which have
been most often used in generating inhalation data given to the
Agency. The whole—body chamber exposures have primary disadvantages
in that the test agent is deposited on the skin of the animal and
may be absorbed directly through the skin and also the aninals
may lick the test material from their fur and receive a significant
oral exposure as well. Nose—only and head—only inhalation exposure
systems prevent oral and dermal exposures. In these systems the
animals are restrained in a tube with their heads or noses extending
into a chamber. The restraining tubes limit movement and may
additionally cause temperature stress to the animals.
The Agency is not recommending whole-body exposures over
alternative exposures, such as nose—only or head—only exposure
systems. However, recent developments suggest that the nose—only
systems (such as those described in Leong 1981, Phalen 1984 and
Salem 1986) will prove to be superior to whole—body exposure
systems.
When alternative systems are used in place of whole-body
chambers, the same exposure parameters discussed above for the
whole-body systems must also be determined and reported. Thus,
the chamber concentrations, particle size, temperature (inside
the mixing chambers as well as those experienced in the constraint
tubes), etc., must be monitored.
17

-------
V. ANIMAL OBSERVATION
A. Routine
Inhalation studies range from the acute short—term
studies to the subchronic, chronic, and special studies noted in
the introduction. The types and extent of the observati ons
depend on the type of inhalation study being carried out. All of
the studies involve daily observations for toxic signs, body
weight determinations, and gross necropsy examinations. The
repeat studies involve the assessment of food consumption, hema-
tology, blood chemistries, urinalysis, organ weights, and histo—
pathologic examination.
The reviewer is referred to the SEPs for guidance in
evaluating subchronic and chronic studies (Paynter et al. 1985),
oncogenicity studies (Paynter 1985), and teratology (Chitlick et
al. 1985). Table 6 lists a number of routine clinical laboratory
tests that should be considered in evaluating repeat inhalation
exposures studies, and Table 7 the gross necropsy and histopatho—
logical examinations that should be considered.
In inhalation studies, special considerations need to
be given to the examination of the nasopharyngeal areas and the
lungs. This involves the gross examination of the nasal areas
and sinuses and the sectioning of these areas for histopathological
examination. The lungs should be removed intact, weighed, and
treated with suitable fixative to ensure that the lung structure
is rn intained. This usually involves the perfusion of the lungs
with fixative under low pressure.
B. Lung Function Studies
Pulmonary function studies are not carried out on a
routine basis and most commercial animal laboratories are not
equipped to perform them. Many pulmonary medical specialists do
carry out such studies on a routine basis when diagnosing and
monitoring human pulmonary disease. These studies measure
diffusional capacities of gases into and out of the lungs, lung
compliance, and changes in lung volumes. These studies are
mentioned here because they are able to detect early functional
deficits caused by toxic materials which are not evident by usual
observations used and they provide quantitative measurements for
pulmonary functional impairment.
18

-------
VI. ACCEPTABILITY OF INHALATION STUDIES
Not infrequently, an inhalation toxicity report will lack
certain information which makes it difficult or impossible to
assess its value. The reviewer is justified in rejecting a study
without further review if:
o The test substance cannot be identified relati Te to its
composition such that one cannot determine the percent
of the active ingredient or the types of contaminants
or biologically active additives that might be involved.
o It is not possible to understand the exposure
conditions——the chamber size, shape and design is not
identified and that it is not possible to determine that
the test atmosphere was uniformly distributed to the
animals within the chamber.
o The actual or analytical concentrations from the
breathing zone of the animal were not measured. Nominal
concentrations by themselves cannot be used as a measure
of animal exposure. This would also be the case if the
methods used to determine the chamber concentrations
were not appropriate for the test substance.
o The test substance is an aerosol (spray or dust), and
the particle size was too large or improperly characterized,
one could not insure the animals were exposed to particles
that could be inhaled and distributed throughout the
pulmonary system. -
In a number of situations, a considerable amount of
judgment must be used in questioning the validity of a study,
especially when the study finds rio adverse effects on the animals
tested. When it is known from other toxicity tests that an agent
is irritating (to the skin, eye, or mucous membranes) or highly
toxic by other routes (oral or dermal), then the animals should
show definite signs of toxicity during inhalation exposures. If
the expected toxic effects are absent, the study may be suspect
as to whether or not the animals in fact were adequately exposed.
A laboratory audit should be requested if all of the other
information appears to be in order.
Beyond these more obvious situations, it may be difficult
for reviewers to spot problems with the inhalation exposure
techniques, unless they have had direct experience with the
equipment and methods used in the study. When questions arise
the reviewer should not hesitate to consult with inhalation
toxicology specialists.
19

-------
REFERENCES
Chitlik, L.D.; Bui, Q.Q.; Burin, G.L.; Dapson, S.C. (1985)
Hazard Evaluation Division. Standard Evaluation Procedure.
Teratology Studies. U.S. Environmental Protection Agency,
Washington, DC, EPA—540/9—85—018.
Davies, C.N., editor (1961) ‘Inhaled Particles and Vapor.’
Proceeding of an International Symposium Organized by the
British Occupational Hygiene Society. Oxford, March 29 and
April 1, 1960. Pergamon Press, New York.
Drew, R.T., editor (1979) Proceedings: Workshop on Inhalation
Chamber Technology; October 16—17, 1978.’ Brookhaven National
Laboratory, Upton, New York.
Environmental Protection Agency (1982) ‘Pesticide Assessment
Guidelines, Subdivision F, Hazard Evaluation: Human and
Domestic Animals.’ U.S. Environmental Protection Agency,
Washington, DC, EPA—540/9—82/025.
Environmental Protection Agency (1984) 40 CFR Part 158. Data
Requirements for Pesticide Registration; Final Rule. FEDERAL
REGISTER 48:42856—42905.
Fraser, D.A.; Bales, R.E.; Lippmanri, M.; Stokinger, H.E. (1957)
‘Exposure Chambers for Research in Animal Inhalation’ U.S.
Department of Health, Education, and Welfare, Public Health
Service. Monograph No. 57.
Gross, S.B. (1981) Regulatory Guidelines for Inhalation
Toxicity Testing, pages 279—298, In ‘Proceedings of the
Inhalation Toxicity and Technology Symposium,’ Ann Arbor
Science Publishers, Inc.
Gross, S.B. (1987) Chapter 13, Issues of Regulatory Requirements
for Inhalation Toxicity Testing. In ‘Inhalation Toxicology,’
Harry Salem, editor. Marcel—Dekker, Publisher, New York.
Leong, B.K.J., editor (1981) ‘Proceedings of the Inhalation
Toxicology and Technology Symposium.’ Kalamazoo, Michigan.
October 23—24, 1980. Ann Arbor Science Publishers, Inc.
McClellan, R.O.; Hobbs, C.H. (1986) Chapter 17, Generation,
Characterization, and Exposure Systems for Test Atmospheres.
Pages 257—284, In ‘Safety Evaluations of Drugs and Chemicals,’
W. Eugene Lloyd, editor. Hemisphere Publishing Corporation,
Washington, DC.
20

-------
Mercer, T.T. (1973) ‘Aerosol Technology in Hazard Evaluation.
Academic Press, New York.
National Academy of Sciences/National Research Council (1972)
‘Institute of Laboratory Animal Resources. Guide for the Care
and Use of Laboratory Animals.” Government Printing Office.
DHEW Publication. Washington, DC..iqu
National Academy of Sciences (1977) Inhalation Exposure.
Chapter 4, pages 60—73, In ‘Princ )le and Procedures for
Evaluating the Toxicity of Housel’i daSubstances. ’ National
Academy of Sciences, Washington, DC.
National Institute for Occupational 1th (1973) ‘The
Industrial Environment——Its Evaluation and Control.’ U.S.
Department of Health, Education, and Welfare, Public Health
Service, Center for Disease Control.
Paynter, O.E.; Harris, J’.E.; Burin, G.J; Jaeger, R.B. (1985)
‘Hazard Evaluation Division. Standard Evaluation Procedure.
Toxicity Potential: Guidance for Analysis and Evaluation of
Subchronic and Chronic Exposure Studies.’ U.S. Environmental
Protection Agency, Washington, DC, EPA-540/9—85—020.
Paynter, O.E. (1985) ‘Hazard Evaluation Division. Standard
Evaluation Procedure. Oncogenicity Potential: Guidance
for Analysis and Evaluation of Long—Term Rodent Studies.’
U.S. Environmental Protection Agency, Washington, DC,
EPA— 540/9—8 5—0 19
Phalen, R.F. (1984) ‘Inhalation Studies: Foundations and
Techniques.’ CRC Press, Inc., Boca Ratoni, FL.
Ryon, M.G. (1984) Inhalation Toxicology. Chapter 4, pages
149—241, In ‘Scientific Rationale for the Selection of Toxi-
city Testing Methods. II. Teratology, Irnrnuniotoxicology, and
Inhalation Toxicology.” Micheal G. Ryon and Daijit S. Sawhney,
eds. ORNL—6094 and EPA—560/6—84-.004. Oak Ridge National
Laboratory, Oak Ridge, TN.
Salem, H., editor (1987) ‘Inhalation Toxicology.’ Marcel—
Dekker, Publisher, New York.
Snipes, M.B.; Oslon, T.R.; Yeh, H.C. (1983) Deposition and
Retention Patterns for 3—, 9—, and 15—urn Latex Microspheres
Inhaled by Rats. Pages 128—132 In “Inhalation Toxicology
Research Institute Annual Report, 1982—83.’ Lovelace Bio-
medical and Environmental Research Institute, Albuquerque,
NM.
21

-------
TABLE 1
OVERVIEW OF ACUTE INHALATION TOXICITY TESTINGa
1. When Required : Potentially significant expr sures to gases,
vapors of liquids or solids, aerosols of tds (sprays) or
solids (dusts), combination of these. -
2. Pur?ose : Determine LC 50 , confidence limi dope; relative
toxLcity for labeling; define target org •d mechanism of
action if possible.
3. Definitions : LC 50 , Aerodynamic vs. Measu Diameters, Median
Diameters, Inhalable Diameters.
4. Standards
Test Substance : Technical, manufacturing, formulation
products, and7or use dilutions; test same physical and
chemical form as expected exposure; must be respirable for
test animal.
Vehicle : If required should be characterized, ideally should
not significantly alter chemical or toxicological properties.
Test Animals : Young adult laboratory rats preferred (125 to
250 gm); other species with justification; males and females
(nulliparous, nonpregnant), five animals/sex/test level.
Limit Test : If five males and five females, exposed for 4
hours to 5 mg/L actual concentration of respirable test
substance unless not achievable because of the characteristics
of the test agent, causes no deaths, no further acute inhala-
tion testing will be necessary.
Controls : None required.
Exposure Levels : Usually four or more levels, a minimum of
three concentrations possible, should bracket the LC 50
(between 10 and 90% mortality), only one 0% and one 100%
adjacent mortality levels should be used to calculate the
LC 50 and slope.
Duration of Exposure : Minimum of single 4—hour, longer if
indicated by expected human exposure; allow added time for
equilibration of chamber.
Taken from S.B. Gross (1987).
T—1

-------
TABLE 1
OVERVIEW OF ACUTE INHALATION TOXICITY TESTING (cont’d)a
Exposure Conditions : Whole—body exposures in dynamic flow
chambers preferred, uniform atmosphere distribution, single
caging preferred, animal volume equal to or less than 5
percent of chamber; alternative methods with approval.
Chamber Measurements
*Air flow : At least 10 volume changes/hour, monitor
frequently, record every 30 minutes.
Temperature : 22 + 2 °C, monitor frequently, record
every 30 minutes.
Humidity : 40 to 60 percent unless altered by generation
of chamber atmosphere, monitor frequently.
Concentrations : Nominal; Analytical (gaseous and/or
particulate) from the breathing zone, times during
run (after equilibrium and during last hour of exposure),
minimum suggested.
Particle Size for Aerosols : Should characterize during
development of the generation system and confirm at least
one time during run depending on the expected variation
within chamber; sampled from breathing zone of animal;
must be respirable for test animal.
Observations : Close observations during and just after
exposures; daily (early morning and late afternoon) for
14 days; record abnormalities (nature, onset, duration);
body weights before exposure, weekly and at sacrifice.
Necropsy : All animals whether dying or sacrificed, desirable.
Histopathology : At option of study director.
5. Test Reports
Summary of Findings
GLP: Facility and personnel information, quality assurance,
storage of samples, records, etc.
Taken from S.B. Gross (1987).
* Constant electronic monitoring desirable.
T-2

-------
TABLE 1
OVERVIEW OF ACUTE INHALATION TOXICITY TESTING (cont ’d)a
General Section Requirements : Test substance identification,
characteristics; animal data; animal care, deviations from
Guideline procedures; statistical methods used; quarity
assurance, etc.
Test Specific Information
Test Substance : Vapor pressure, boiling point,
flammability, exp]osivity, and stability.
Exposure Chambers : Design, operation, make—up air, waste
disposal, equipment for controlling temperature, humidity,
the generating system, methods for analytical concentrations,
particle sizing, etc.
Operating Data : Indiv.idual and summarized (means SD’s)——
Air flow, chamber temperature and humidity, nominal
concentrations (without SD’S), analytical concentrations,
and for aerosols, median particle sizes and geometric
SD’s specifying the percent of respirable and nonrespirable
based on weight.
Limit Test Results : When applicable.
Response Data
Toxicity observed, onset, duration, animals involved,
time to death;
Body weights;
Necropsy findings; and
LC5O, 95% confidence limits and slope of the dose—
mortality curve for each sex, method of calculation.
References for methods and equipment used.
Discussion and Evaluation
a Taken from S.B. Gross (1987).
T- 3

-------
TABLE 2
OVERVIEW OF 90-DAY INHALATION TOXICITY TESTINGa
1. When Required : Repeat exposures to products containing
Inhalable chemicals (as gases, vapors, aerosols, or
combinations).
2. Purpose : Provides information on the types chemically
induced toxicity and establishes a no-observed—effect level
(NOEL.) under the test conditions; used in determining
allowable exposure, mechanisms of toxicity.
3. Definitions : Same as acute inhalation toxicity testing.
4. Standards
Test Substance : Technical grade of the active ingredient(s).
Vehicle : If required should be.characterized; ideally should
not significantly alter chemical or toxicological properties
of test substance.
Test Animals : Young adult laboratory rats preferred (125 to
250 gm); other test species with justification; start with
enough animals (15 to 20 sex/level) so as to have at least 10
animals/sex/level at end of experiment for clinical testing,
necropsy and histology; add additional animals if interim
sacrif ices or recovery groups are planned.
Exposure Concentrations : At least three levels plus
controls, concurrently. High level——definite signs of
toxicity. Low level——no toxicity (responses similar to
controls). Intermediate level(s).
Control : One vehicle or chamber (air) control; air and
vehicle control with use of uncharacterized vehicles.
Duration of Exposure : 6 hours/day, 5 days/week; extend
exposure if indicated by exposure conditions.
Exposure Conditions : Whole—body in dynamic flow chambers
preferred, uniform air distribution, animal volume equal to
or less than 5 percent, single caging preferred; alternative
methods with approval.
a Taken from S.B. Gross (1987).
T- 4

-------
TABLE 2
OVERVIEW OF 90-DAY INHALATION TOXICITY TESTING (cont’d)a
Chamber Measurements
*Air Flow : at least 10 changes per hour, adequate
oxygen (19% or more), etc.; record every hour.
*Temperature : 22 ± 2 °C, record every hour.
Humidity : 40 to 60 percent unless altered by generation
of atmosphere.
Concentrations : Nominal for each run; analytical (gaseous
and7or particulate), sampled at breathing zone at least
once after equilibrium.
Particle Size for Aerosols : Sampled at breathing zone; at
least one sample per run desirable; must be respirable for
test animal.
Observations : Daily (early morning and late afternoon),
body weights weekly; food consumption if indicated.
Clinical Laboratory TestinO
Ophthalrnologlcal examination at end of study on at least
high level and control groups, other groups with positive
findings.
Hematology routine on all animals at end of study; specific
tests as indicated by observed or expected toxicity.
Blood Chemistry routine on all animals at end of study,
appropriate to evaluate electrolyte balance, carbohydrate
metabolism, liver and kidney function; pulmonary function
and other tests as indicated by observed or expected
toxicity.
Urinalysis routine on nonrodent species; on rodents, only
as indicated based on observed or expected toxicity.
Gross Necropsy : Complete necropsy on all animals; organ
weights on liver, kidney, adrenals, and testes in all
animals; preserve organs and all lesions for possible future
histopathological examination; perfusion of lungs with
fixative considered desirable.
a Taken from S.B. Gross (1987).
* Constant electronic monitoring desirable.
T-5

-------
TABLE 2
OVERVIEW OF 90-DAY INHALATION TOXICITY TESTING Ccont’d)a
Histopathology : Examine microscopically all gross lesions;
completehistopathology for high level and control animals;
for the intermediate and low level groups, examine screen
organs (liver, kidney, and heart), target organs and organs
with positive findings from the high level group and the
controls. Full histopathology of the respiratory tract
on all test groups.
5. Test Report
Summary
GLP and General Section Requirements
Test Specific Information
Test Substance : Vapor pressure, boiling point,
flammability, explosivity, and stability.
Exposure Chambers : Description, operation, etc.
Chamber Operation Data : Tabulation and summary by test
groups: Air flow, temperature, humidity, nominal concen-
trations, analytical concentrations, particle size
measurements-—percent respirable and nonrespirable based on
weight.
Response Data : Tabluations and summaries by test groups:
Toxicity observed, onset, duration, animals involved;
Body weights;
Food consumption if obtained;
Clinical laboratory findings;
Gross and microscopic findings; and
NOEL.
References
Discussion and Evaluation
a Taken from S.B. Gross (1987).
T- 6

-------
TABLE 3
SOME PHYSIOLOGICAL INDICES OP MAIl AND ANIMALSa
Guinea
Physiological Index Man Dog Cat Rabbit Pig Rat Mouse
Body surface (in 2 ) 1.8 0.528 0.2 0.18 0.040 0.030 0.006
Relation body surface to 0.0257 0.044 0.066 0.072 0.12 0.15 0.3
body weight (m 2 /kg)
Basal metabolism (kJ/kg) 105 222 NGb 188 360 615 711
Frequency of respiration 14—18 10—30 20—30 50—100 80—135 110—135 140—210
(sin)
Size of alveoli (urn) 150 100 100 HG HG 50 30
Surface of lungs (in 2 ) 50 100 7.2 5,21 1.47 0.56 0.12
Relation of lung surface 0.7 8.3 2.8 2.5 3.2 3.3 5.4
to body weight ( 1n 2 /kg)
Inhaled air (eL) 616 40—60 HG HG 1.75 0.865 0.154
Lung ventilation (mi./min) 8732 HG 1000 600 155 73 25
Relation of lung ventilation 0.13 HG 0.30 0.29 0.33 0.365 1.24
to body weight (mL/min/g)
Consumption of oxygen 203.1 3600 9420 522.7 2180 2199 3910
(eL/kg/h)
Elimination of CO 2 (niL/kg/h) 168.8 HG HG HG N C 2650 4240
Coefficient of respiration 0.82 HG HG 0.83 HG 0.82 0.85—1.33
Pulse frequency for I elm 70—72 90—1 20 120—180 150—240 206—280 300—500 520—780
Taken from Ryon, (1984) with permission from the editors.
b HG not given.

-------
TABLE 4
OUTPUT CHARACTERISTICS OP SOME CO?4PRESS D AIR NEBULIZERS
Mass
Nebulizer
Jet
Pressure
(psi)
Median
Diameter
(urn)
Geometric
Standard
Deviation
Specific Output
(uL Solution/L
jet air)
Output (uL
solution/nina)
Reference
Vaponefrin
12
5.6
1.80
—29
117
Mercer et al.
1965
DeVilbiss #40
10
4.1
—1.85
16 • 0 b
155
Mercer et al.
1968b
DeVilbiss #40
20
3.2
— .1.85
13 • 8 b
229
Mercer et al.
1968b
DeVilbiss #40
30
2.8
—1.85
12 • 8 b
270
Mercer et al.
1968b
Bennett Twin (2814)
7.5
6.8
1.80
23.8
119
Mercer et al.
1965
Puritan (R6—051)
23
6.5
1.90
26.6
266
Mercer et al
1965
Lauterbach
10
3.8
—2.05
3.9
30 c
Mercer et al.
1968b
Lauterbach
20
2.4
—2.05
5.7
6 7c
Mercer et al.
1968b
Lauterbach
30
2.4
—2.05
6.0
91
Mercer et al.
1968b
Dautrebande D—30
10
1.7
—1.65
1.42
21
Mercer et al.
1968b
Dautrebande 0—30
20
1.4
—1.65
2.3
49
Mercer et al.
1968b
Dautrebande 0—30
30
1.3
-1.65
2.4
65
Mercer et al.
1968b
Lovelace
10
NGd
15.3
14 e
Mercer et al.
1968b
Lovelace
20
5.4
1.90
30
39
Mercer et al.
1968b
Lovelace
30
NG
NG
35
58 • 3 e
Mercer et al.
1968b
Collison
15
NC
NG
8.7
53
May 1973
Collison
25
1.9
2.5
6.7
55
May 1973
Collison
30
NC
NG
5.8
55
May 1973
Retec
20
5.7
1.8
35.2
208
Raabe 1972
Retec
30
3.6
2.0
35.9
284
Raabe 1972
Retec
50
3.2
2.2
319
376
Raabe 1972
a Dioctyl sebacate is the nebulized liquid for the Colilson,
dye ts the nebulized liquid for the other nebulizers.
b Zer6 auxiliary air flow.
C Orifice diameter, 0.032 in.
d NG = not given.
e Orifice diameter, 0.001 in.
while water containing salt or a fluorescent
I
Source: Ryan (1984) with permission of the editors.

-------
TABLE 5
SAMPLE PARTICLE SIZE DATAa
Effective Cutoff
Diameter Deposit Cumulative
Stage ( Lnicrou etera) Conc (10 6 M) Percent e Perøentage
1 5.465 0.00 0.000 100.000
2 3.360 0.00 0.000 100.000
3 2.364 3.00 0.265 100.00
4 1.817 16.00 1.415 99.735
5 1.204 260.00 22.989 99.320
6 0.828 540.00 47.745 75.332
7 0.373 310.00 27.410 27.587
8 0.000 2.00 0.177 0.177
a Taken from a laboratory experiment.
T- 9

-------
TABLE 6
ROUTINE CLINICAL LABORATORY TESTSa
1 ematology : Hernatocrit, hemoglobin, erythrocyte count, total and
differential leukocyte counts, clotting potential such as
clotting time, prothrombin time, thromboplastin time-or platelet
count.
Blood Chemistry : Measure of electrolyte balance, carbohydrate
metabolism liver and kidney function such as calcium, phosphorus,
chlorine, sodium, potassium, fasting glucose, lactic dehydro—
genase, serum glutamic—pyruvic transaminase, serum glutamic
oxloacetic transaminase, serum alkaline phosphatase, urea
nitrogen, albumin, blood creatinine, total bilirubin, and
total protein. Might consider analyses of lipid, hormones,
acid/base balance and carboxyhemoglobin.
Urinalysis : Volume, color, specific gravity or osmolarity, pH,
protein, glucose, ketones, formed elements (RBC’s, WBC’s,
epithelial cells, etc.), casts, crystalline and amorphous
materials, and blood pigments.
Taken from S.B. Gross (1987).
T- 10

-------
TABLE 7
COMPLETE GROSS NECROPSY AND HISTOPATHOLOGICAL EXAMINATIONSa
Grossly examine external surface of the body, all orifices and
— the cranial, thoracic and abdominal cavities and their contents.
Grossly and histologically examine following organs: Brain (at
least 3 levels), spinal cord (at least 2 levels), pituitary,
salivary glands, thymus, thyroid/parathyroid, heart, aorta,
esophagus, lung (with mainstem bronchi), trachea, liver, stomach,
small and large intestines, spleen, kidneys, adrenals, pancreas,
urinary bladder, gonads, accessory genital organs, mammary gland,
skeletal muscle, lymph node, sternum with bone marrow, and
peripheral nerve, skin for dermal tests.
a Taken from S.B. Gross (1987).
T-ll

-------
FIGUIRE 1. Components of a simplified chamber exposure system
(Adapted from R.F. Phafen, 1984 with permission from the author)
aagiEWt AIR
M itt
SI ATMINQ za t
CMARAC I ER IflY ION
ATM MERI

-------
FIGURE 2. Schematic diagram of the Rochester exposure chamber
Taken from Drew (1979)
( NAUST
AIR
—r- 3

-------
FIGURE 3._Schematic diagram of the New York University
exposure chamber
Taken from Drew (1979)
EXHAUST AIR
SUPPLY AIR CONTAMIraANT
SUPPLY

-------
FIGURE 4.
Equalibrations of Exposure Chamber:
Concentration-time relationships in a chamber operated for a long period of time,
ta Start of flow through chamber; t —time at which equilibrium concentration
is reached; tb — time at which test agent is no longer added to airflow; and t end
of airflow through chamber. (Taken from Ryon, 1984).
z
0
I—
z
w
0
z
0
0
0
ta
t 199 tt tc
TIME

-------
FIGURE 5. Diagram of the DeVilbiss nebulizer
LIQUID INLET
TUBE
VENT
COMPRESSED AIR
IN

-------
FIGURE 6. Aerodynamic size (i Diameter) vs. Dieposition in the Lung
—DIFFUSlON .
‘ —IMPACT1ON-
-4
- SEDIMENTATION,
100
80
C
0
I
$A YNQEA
Aerodynamic Size (urn)

-------
FIGURE 7. Schematic view of cascade impactor
COVER PLATE
COVER SLIP
— 0 RING
VER SLIP
JP9ORr
CLE
JPPORT WRING
HOUSING
JET COLLECTOR SPACER
MEMBRANE FILTER
VACUUM APPLIED

-------
FIGURE 8. Example particle size graph:
Aerodynamic diameter - 0.97 urn
Geometric standard deviation - °“. ! ! - 1.37
50% 0.97
E
2
4-.
E
C,
0 1.0
U
E
C,
C
>.
0
0
C)
0.0I 0.10.2 0.6 I 3 3 10 20 30401060 70 60 90 9060 g9 909 99.90
Curnutative Percentage Less Than Stated Size

-------