[Code of Federal Regulations]
[Title 40, Volume 14]
[Revised as of July 1, 2003]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR79.61]

[Page 541-551]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 79--REGISTRATION OF FUELS AND FUEL ADDITIVES--Table of Contents
 
            Subpart F--Testing Requirements for Registration
 
Sec. 79.61  Vehicle emissions inhalation exposure guideline.

    (a) Purpose. This guideline provides additional information on 
methodologies required to conduct health effects tests involving 
inhalation exposures to vehicle combustion emissions from fuels or fuel/
additive mixtures. Where this guideline and the other health effects 
testing guidelines in 40 CFR 79.62 through 79.68 specify differing 
values

[[Page 542]]

for the same test parameter, the specifications in the individual health 
test guideline shall prevail for that health effect endpoint.
    (b) Definitions. For the purposes of this section the following 
definitions apply.
    Acute inhalation study means a short-term toxicity test 
characterized by a single exposure by inhalation over a short period of 
time (at least 4 hours and less than 24 hours), followed by at least 14 
days of observation.
    Aerodynamic diameter means the diameter of a sphere of unit density 
that has the same settling velocity as the particle of the test 
substance. It is used to compare particles of different sizes, densities 
and shapes, and to predict where in the respiratory tract such particles 
may be deposited. It applies to the size of aerosol particles.
    Chronic inhalation study means a prolonged and repeated exposure by 
inhalation for the life span of the test animal; technically, two years 
in the rat.
    Concentration means an exposure level. Exposure is expressed as 
weight or volume of test aerosol/substance per volume of air, usually 
mg/m3 or as parts per million (ppm) over a given time period. 
Micrograms per cubic meter ([mu]g/m3) or parts per billion 
may be appropriate, as well.
    Cumulative toxicity means the adverse effects of repeated exposures 
occurring as a result of prolonged action or increased concentration of 
the administered test substance or its metabolites in the susceptible 
tissues.
    Inhalable diameter means 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 anywhere within the respiratory tract from the trachea to the 
alveoli.
    Mass median aerodynamic diameter (MMAD) means the calculated 
aerodynamic diameter, which divides the particles of an aerosol in half 
based on the mass of the particles. Fifty percent of the particles in 
mass will be larger than the median diameter, and fifty percent will be 
smaller than the median diameter. MMAD describes the particle 
distribution of any aerosol based on the weight and size of the 
particles. MMAD and the geometric standard deviation describe the 
particle-size distribution.
    Material safety data sheet (MSDS) means documentation or information 
on the physical, chemical, and hazardous characteristics of a given 
chemical, usually provided by the product's manufacturer.
    Reynolds number means a dimensionless number that is proportional to 
the ratio of inertial forces to frictional forces acting on a fluid. It 
quantitatively provides a measure of whether flow is laminar or 
turbulent. A fluid traveling through a pipe is fully developed into a 
laminar flow for a Reynolds number less than 2000, and fully developed 
into a turbulent flow for a Reynolds number greater than 4000.
    Subacute inhalation toxicity means the adverse effects occurring as 
a result of the repeated daily exposure of experimental animals to a 
chemical by inhalation for part (less than 10 percent) of a lifespan; 
generally, less than 90 days.
    Subchronic inhalation study means a repeated exposure by inhalation 
for part (approximately 10 percent) of a life span of the exposed test 
animal.
    Toxic effect means an adverse change in the structure or function of 
an experimental animal as a result of exposure to a chemical substance.
    (c) Principles and design criteria of inhalation exposure systems. 
Proper conduct of inhalation toxicity studies of the emissions of fuels 
and additive/fuel mixtures requires that the exposure system be designed 
to ensure the controlled generation of the exposure atmosphere, the 
adequate dilution of the test emissions, delivery of the diluted 
exposure atmosphere to the test animals, and use of appropriate exposure 
chamber systems selected to meet criteria for a given exposure study.
    (1) Emissions generation. Emissions shall be generated according to 
the specifications in 40 CFR 79.57.
    (2) Dilution and delivery systems. (i) The delivery system is the 
means used to transport the emissions from the generation system to the 
exposure system. The dilution system is generally a component of the 
delivery system.
    (ii) Dilution provides control of the emissions concentration 
delivered to

[[Page 543]]

the exposure system, serving the function of diluting the associated 
combustion gases, such as carbon monoxide, carbon dioxide, nitrogen 
oxides, sulfur dioxide and other noxious gases and vapors, to levels 
that will ensure that there are no significant or measurable responses 
in the test animals as a result of exposure to the combustion gases. The 
formation of particle species is strongly dependent on the dilution 
rate, as well.
    (iii) The engine exhaust system shall connect to the first-stage-
dilution section at 90 deg. to the axis of the dilution section. This is 
then connected to a right angle elbow on the center line of the dilution 
section. Engine emissions are injected through the elbow so that exhaust 
flow is concurrent to dilution flow.
    (iv) Materials. In designing the dilution and delivery systems, the 
use of plastic, e.g., PVC and similar materials, copper, brass, and 
aluminum pipe and tubing shall be avoided if there exists a possibility 
of chemical reaction occurring between emissions and tubing. Stainless 
steel pipe and tubing is recommended as the best choice for most 
emission dilution and delivery applications, although glass and teflon 
may be appropriate, as well.
    (v) Flow requirements. (A) Conduit for dilute raw emissions shall be 
of such dimensions as to provide residence times for the emissions on 
the order of less than one second to several seconds before the 
emissions are further diluted and introduced to the test chambers. With 
the high flow rates in the dilute raw emissions conduit, it will be 
necessary to sample various portions of the dilute emissions for 
delivering differing concentrations to the test chambers. The unused 
portions of the emissions stream are normally exhausted to the 
atmosphere outside of the exposure facility.
    (B) Dimensions of the dilute raw exhaust conduit shall be such that, 
at a minimum, the flow Reynolds number is 70,000 or greater (see Mokler, 
et al., 1984 in paragraph (f)(13) of this section). This will maintain 
highly turbulent flow conditions so that there is more complete mixing 
of the exhaust emissions.
    (C) Wall losses. The delivery system shall be designed to minimize 
wall losses. This can be done by sizing the tubing or pipe to maintain 
laminar flow of the diluted emissions to the exposure chamber. A flow 
Reynolds number of 1000-3000 will ensure minimal wall losses. Also, the 
length of and number and degree of bends in the delivery lines to the 
exposure chamber system shall be minimized.
    (D) Whole-body exposure vs. nose-only exposure delivery systems. 
Flow rates through whole-body chamber systems are of the order of 100 
liters per minute to 500 liters per minute. Nose-only systems are on the 
order of less than 50 liters per minute. To maintain laminar flow 
conditions, the principles described in paragraph (c)(2)(v)(C) of this 
section apply to both systems.
    (vi) Dilution requirements. (A) To maintain the water vapor, and 
dissolved organic compounds, in the raw exhaust emissions stream, a 
manufacturer/tester will initially dilute one part emissions with a 
minimum of five parts clean, filtered air (see Hinners, et al., 1979 in 
paragraph (f)(11) of this section). Depending on the water vapor content 
of a particular fuel/additive mixture's combustion emissions and the 
humidity of the dilution air, initial exhaust dilutions as high as 1:15 
or 1:20 may be necessary to maintain the general character of the 
exhaust as it cools, e.g., M100. At this point, it is expected that the 
exhaust stream would be further diluted to more appropriate levels for 
rodent health effects testing.
    (B) A maximum concentration (minimum dilution) of the raw exhaust 
going into the test animal cages is anticipated to lie in the range 
between 1:5 and 1:50 exhaust emissions to clean, filtered air. The 
minimum concentration (maximum dilution) of raw exhaust for health 
effects testing is anticipated to be in range between 1:100 and 1:150. 
Individual manufacturers will treat these ranges as approximations only 
and will determine the optimum range of emission concentrations to 
elicit effects in Tier 2 health testing for their particular fuel/fuel 
additive mixture.
    (3) Exposure chamber systems--(i) Referenced Guidelines. (A) The 
U.S. Department of Health and Human Services

[[Page 544]]

``Guide for the Care and Use of Laboratory Animals'' (Guide), 1985 cited 
in paragraph (c)(3)(ii)(A)(4), and in paragraphs (d)(2)(i), (d)(2)(ii), 
(d)(2)(iii), (d)(4)(ii), and (d)(4)(iii) of this section, has been 
incorporated by reference.
    (B) This incorporation by reference was approved by the Director of 
the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 
51. Copies may be purchased from the Superintendent of Documents, U.S. 
Government Printing Office, Washington, DC 20402. Copies may be 
inspected at U.S. EPA, OAR, 401 M Street SW, Washington, DC 20460 or at 
the Office of the Federal Register, 800 North Capitol Street NW., suite 
700, Washington, DC.
    (ii) Exposure chambers. There are two basic types of dynamic 
inhalation exposure chambers, whole-body chambers and nose-/head-only 
exposure chambers (see Cheng and Moss, 1989 in paragraph (f)(8) of this 
section).
    (A) Whole-body chambers. (1) The flow rate through a chamber shall 
be maintained at 15 air changes per hour.
    (2) The chambers are usually maintained at a slightly negative 
pressure (0.5 to 1.5 inch of water) to prevent leakage of test substance 
into the exposure room.
    (3) The exposure chamber shall be designed in such a way as to 
provide uniform distribution of exposure concentrations in all 
compartments (see Cheng et al., 1989 in paragraph (f)(7) of this 
section).
    (4) Animals are housed in separate compartments inside the chamber, 
where the whole surface area of an animal is exposed to the test 
material. The spaces required for different animal species shall follow 
the Guide. In general, the volume of animal bodies occupy less than 5 
percent of the chamber volume.
    (B) Head/nose-only exposure chambers. (1) In head/nose-only exposure 
chambers, only the head (oronasal) portion of the animal is exposed to 
the test material.
    (2) The chamber volume and flow rates are much less than in the 
whole-body exposure chambers because the subjects are usually restrained 
in a tube holder where the animal's breathing can be easily monitored. 
The head/nose-only exposure chamber is suitable for short-term exposures 
or when use of a small amount of test material is required.
    (iii) Since whole-body exposure appears to be the least stressful 
mode of exposure, it is the preferred method. In general, head/nose only 
exposure, which is sometimes used to avoid concurrent exposure by the 
dermal or oral routes, i.e., grooming, is not recommended because of the 
stress accompanying the restraining of the animals. However, there may 
be specific instances where it may be more appropriate than whole-body 
exposure. The tester shall provide justification for its selection.
    (d) Inhalation exposure procedures--(1) Animal selection. (i) The 
rat is the preferred species for vehicle emission inhalation health 
effects testing. Commonly used laboratory strains shall be used. Any 
rodent species may be used, but the tester shall provide justification 
for the choice of that species.
    (ii) Young adult animals, approximately ten weeks of age for the 
rat, shall be used. At the commencement of the study, the weight 
variation of animals used shall not exceed 20 percent of the 
mean weight for each sex. Animals shall be randomly assigned to 
treatment and control groups according to their weight.
    (iii) An equal number of male and female rodents shall be used at 
each concentration level. Situations may arise where use of a single sex 
may be appropriate. Females, in general, shall be nulliparous and 
nonpregnant.
    (iv) The number of animals used at each concentration level and in 
the control group(s) depends on the type of study, number of biological 
end points used in the toxicity evaluation, the pre-determined 
sensitivity of detection and power of significance of the study, and the 
animal species. For an acute study, at least five animals of each sex 
shall be used in each test group. For both the subacute and subchronic 
studies, at least 10 rodents of each sex shall be used in each test 
group. For a chronic study, at least 20 male and 20 female rodents shall 
be used in each test group.
    (A) If interim sacrifices are planned, the number of animals shall 
be increased by the number of animals

[[Page 545]]

scheduled to be sacrificed during the course of the study.
    (B) For a chronic study, the number of animals at the termination of 
the study must be adequate for a meaningful and valid statistical 
evaluation of chronic effects.
    (v) A concurrent control group is required. This group shall be 
exposed to clean, filtered air under conditions identical to those used 
for the group exposed to the test atmosphere.
    (vi) The same species/strain shall be used to make comparisons 
between fuel-only and fuel/additive mixture studies. If another species/
strain is used, the tester shall provide justification for its 
selection.
    (2) Animal handling and care. (i) A key element in the conduct of 
inhalation exposure studies is the proper handling and care of the test 
animal population. Therefore, the exposure conditions must conform 
strictly with the conditions for housing and animal care and use set 
forth in the Guide.
    (ii) In whole-body exposure chambers, animals shall be housed in 
individual caging. The minimum cage size per animal will be in 
accordance with instructions set forth in the Guide.
    (iii) Chambers shall be cleaned and maintained in accordance with 
recommendations and schedules set forth in the Guide.
    (A) Observations shall be made daily with appropriate actions taken 
to minimize loss of animals to the study (e.g., necropsy or 
refrigeration of animals found dead and isolation or sacrifice of weak 
or moribund animals). Exposure systems using head/nose-only exposure 
chambers require no special daily chamber maintenance. Chambers shall be 
inspected to ensure that they are clean, and that there are no 
obstructions in the chamber which would restrict air flow to the 
animals. Whole-body exposure chambers will be inspected on a minimum of 
twice daily, once before exposures and once after exposures.
    (B) Signs of toxicity shall be recorded as they are observed, 
including the time of onset, degree, and duration.
    (C) Cage-side observations shall include, but are not limited to: 
changes in skin, fur, eye and mucous membranes, respiratory, autonomic, 
and central nervous systems, somatomotor activity, and behavioral 
patterns. Particular attention shall be directed to observation of 
tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma.
    (iv) Food and water will be withheld from animals for head/nose-only 
exposure systems. For whole-body-exposure systems, water only may be 
provided. When the exposure generation system is not operating, food 
will be available ad libitum. During operation of the generation system, 
food will be withheld to avoid possible contamination by emissions.
    (v) At the end of the study period, all survivors in the main study 
population shall be sacrificed. Moribund animals shall be removed and 
sacrificed when observed.
    (3) Concentration levels and selection. (i) In acute and subacute 
toxicity tests, at least three exposure concentrations and a control 
group shall be used and spaced appropriately to produce test groups with 
a range of toxic effects and mortality rates. The data shall be 
sufficient to produce a concentration-response curve and permit an 
acceptable estimation of the median lethal concentration.
    (ii) In subchronic and chronic toxicity tests, testers shall use at 
least three different concentration levels, with a control exposure 
group, to determine a concentration-response relationship. 
Concentrations shall be spaced appropriately to produce test groups with 
a range of toxic effects. The concentration-response data may also be 
sufficient to determine a NOAEL, unless the result of a limit test 
precludes such findings. The criteria for selecting concentration levels 
has been published (40 CFR 798.2450 and 798.3260).
    (A) The highest concentration shall result in toxic effects but not 
produce an incidence of fatalities which would prevent a meaningful 
evaluation of the study.
    (B) The lowest concentration shall not produce toxic effects which 
are directly attributable to the test exposure. Where there is a useful 
estimation of human exposure, the lowest concentration shall exceed 
this.

[[Page 546]]

    (C) The intermediate concentration level(s) shall produce minimal 
observable toxic effects. If more than one intermediate concentration 
level is used, the concentrations shall be spaced to produce a gradation 
of toxic effects.
    (D) In the low, intermediate, and control exposure groups, the 
incidence of fatalities shall be low to absent, so as not to preclude a 
meaningful evaluation of the results.
    (4) Exposure chamber environmental conditions. The following 
environmental conditions in the exposure chamber are critical to the 
maintenance of the test animals: flow; temperature; relative humidity; 
lighting; and noise.
    (i) Filtered and conditioned air shall be used during exposure, to 
dilute the exhaust emissions, and during non- exposure periods to 
maintain environmental conditions that are free of trace gases, dusts, 
and microorganisms on the test animals. Twelve to fifteen air changes 
per hour will be provided at all times to whole-body-exposure chambers. 
The minimum air flow rate for head/nose-only exposure chambers will be a 
function of the number of animals and the average minute volume of the 
animals:

Qminimum(L/min)=2 x number of animals x average minute volume

(see Cheng and Moss, 1989 in paragraph (f)(8) of this section).
    (ii) Recommended ranges of temperature for various species are given 
in the Guide. The recommended temperature ranges will be used for 
establishing temperature conditions of whole-body- exposure chambers. 
For rodents in whole-body-exposure chambers, the recommended temperature 
is 22  deg.C  2  deg.C and for rabbits, it is 20  deg.C 
 3  deg.C. Temperature ranges have not been established for 
head/nose-only tubes; however, recommended maximum temperature limits 
have been established at the Inhalation Toxicology Research Institute 
(see Barr, 1988 in paragraph (f)(1) of this section). Maximum 
temperature for rats and mice in head/nose-only tubes is 23  deg.C.
    (iii) Relative humidity. The relative humidity in the chamber air is 
important for heat balance and shall be maintained between 40 percent 
and 60 percent, but in certain instances, this may not be practicable. 
Testers shall follow Guide recommends for a 30 percent to 70 percent 
relative humidity range for rodents in exposure chambers.
    (iv) Lighting. Light intensity of 30 foot candles at 3 ft. from the 
floor of the exposure facility is recommended (see Rao, 1986 in 
paragraph (f)(16) of this section).
    (5) Exposure conditions. Unless precluded by the requirements of a 
particular test protocol, animal subjects shall be exposed to the test 
atmosphere based on a nominal 5-day-per-week regimen, subject to the 
following rules:
    (i) Each daily exposure must be at least 6 hours plus the time 
necessary to build the chamber atmosphere to 90 percent of the target 
exposure atmosphere. Interruptions of daily exposures caused by 
technical difficulties, if infrequent in occurrence and limited in 
duration, may be made up the same day by adding equivalent exposure time 
after the technical problem has been corrected and the exposure 
atmosphere restored to the required level.
    (ii) Normally, no more than two non-exposure days may occur 
consecutively during the test period. However, if a third consecutive 
non-exposure day should occur due to circumstances beyond the tester's 
control, it may be remedied by adding a supplementary exposure day. 
Federal and other holidays do not constitute such circumstances. 
Whenever possible, a make-up day should be taken at the first 
opportunity, i.e., on the next day which would otherwise have been an 
intentional non-exposure day. If a compensatory day must be scheduled at 
the end of the standard test period, then it may occur either:
    (A) Immediately following the last standard exposure day, with no 
intervening non-exposure days; or
    (B) With up to two intervening non-exposure days, provided that no 
fewer than two consecutive compensatory exposure days are completed 
before the test is terminated and the animals sacrificed.
    (iii) Except as allowed in paragraph (d)(5)(ii)(B) of this section, 
in no case shall there be fewer than four exposure days per week at any 
time during the test period.

[[Page 547]]

    (iv) A nominal 90-day (13-week) subchronic test period shall include 
no fewer than 63 total exposure days.
    (6) Exposure atmosphere. (i) The exposure atmosphere shall be held 
as constant as is practicable and must be monitored continuously or 
intermittently, depending on the method of analysis, to ensure that 
exposure levels are at the target values or within stated limits during 
the exposure period. Sampling methodology will be determined based on 
the type of generation system and the type of exposure chamber system 
specified for the exposure study.
    (A) Integrated samples of test atmosphere aerosol shall be taken 
daily during the exposure period from a single representative sample 
port in the chamber near the breathing zone of the animals. Gas samples 
shall be taken daily to determine concentrations (ppm) of the major 
vapor components of the test atmosphere including CO, CO2, 
NOX, SO2, and total hydrocarbons.
    (B) To ensure that animals in different locations of the chamber 
receive a similar exposure atmosphere, distribution of an aerosol or 
vapor concentration in exposure chambers can be determined without 
animals during the developmental phase of the study, or it can be 
determined with animals early in the study. For head/nose-only exposure 
chambers, it may not be possible to monitor the chamber distribution 
during the exposure, because the exposure port contains the animal.
    (C) During the development of the emissions generation system, 
particle size analysis shall be performed to establish the stability of 
an aerosol concentration with respect to particle size. Over the course 
of the exposure, analysis shall be conducted as often as is necessary to 
determine the consistency of particle size distribution.
    (D) Chamber rise and fall times. The rise time required for the 
exposure concentration to reach 90 percent of the stable concentration 
after the generator is turned on, and the fall time when the chamber 
concentration decreases to 10 percent of the stable concentration after 
the generation system is stopped shall be determined in the 
developmental phase of the study. Time-integrated samples collected for 
calculating exposure concentrations shall be taken after the rise time. 
The daily exposure time is exclusive of the rise or the fall time.
    (ii) Instrumentation used for a given study will be determined based 
on the type of generation system and the type of exposure chamber system 
specified for the exposure study.
    (A) For exhaust studies, combustion gases shall be sampled by 
collecting exposure air in bags and then analyzing the collected air 
sample to determine major components of the combustion gas using gas 
analyzers. Exposure chambers can also be connected to gas analyzers 
directly by using sampling lines and switching valves. Samples can be 
taken more frequently using the latter method. Aerosol instruments, such 
as photometers, or time-integrated gravimetric determination may be used 
to determine the stability of any aerosol concentration in the chamber.
    (B) For evaporative emission studies, concentration of fuel vapors 
can usually be determined by using a gas chromatograph (GC) and/or 
infrared (IR) spectrometry. Grab samples for intermittent sampling can 
be taken from the chamber by using bubble samplers with the appropriate 
solvent to collect the vapors, or by collecting a small volume of air in 
a syringe. Intermediate or continuous monitoring of the chamber 
concentration is also possible by connecting the chamber with a GC or IR 
detector.
    (7) Monitoring chamber environmental conditions may be performed by 
a computer system or by exposure system operating personnel.
    (i) The flow-metering device used for the exposure chambers must be 
a continuous monitoring device, and actual flow measurements must be 
recorded at least every 30 minutes. Accuracy must be 5 
percent of full scale range. Measurement of air flow through the 
exposure chamber may be accomplished using any device that has 
sufficient range to accurately measure the air flow for the given 
chamber. Types of flow metering devices include rotameters, orifice 
meters, venturi meters, critical orifices, and turbinemeters (see 
Benedict, 1984 in

[[Page 548]]

paragraph (f)(4) and Spitzer, 1984 in paragraph (f)(17) of this 
section).
    (ii) Pressure. Pressure measurement may be accomplished using 
manometers, electronic pressure transducers, magnehelics, or similar 
devices (see Gillum, 1982 in paragraph (f)(10) of this section). 
Accuracy of the pressure device must be 5 percent of full 
scale range. Pressure measurements must be continuous and recorded at 
least every 30 minutes.
    (iii) Temperature. The temperature of exposure chambers must be 
monitored continuously and recorded at least every 30 minutes. 
Temperature may be measured using thermometers, RTD's, thermocouples, 
thermistors, or other devices (see Benedict, 1984 in paragraph (f)(4) of 
this section). It is necessary to incorporate an alarm system into the 
temperature monitoring system. The exposure operators must be notified 
by the alarm system when the chamber temperature exceeds 26.7  deg.C (80 
 deg.F). The exposure must be discontinued and emergency procedures 
enacted to immediately reduce temperatures or remove test animals from 
high temperature environment when chamber temperatures exceed 29  deg.C. 
Accuracy of the temperature monitoring device will be 1 
deg.C for the temperature range of 20-30  deg.C.
    (iv) Relative humidity. The relative humidity of exposure chambers 
must be monitored continuously and recorded at least every 30 minutes. 
Relative humidity may be measured using various devices (see Chaddock, 
1985 in paragraph (f)(6) of this section).
    (v) Lighting shall be measured quarterly, or once at the beginning, 
middle, and end of the study for shorter studies.
    (vi) Noise level in the exposure chamber(s) shall be measured 
quarterly, or once at the beginning, middle, and end of the study for 
shorter studies.
    (vii) Oxygen content is critical, especially in nose-only chamber 
systems, and shall be greater than or equal to 19 percent in the test 
cages. An oxygen sensor shall be located at a single position in the 
test chamber and a lower alarm limit of 18 percent shall be used to 
activate an alarm system.
    (8) Safety procedures and requirements. In the case of potentially 
explosive test substance concentrations, care shall be taken to avoid 
generating explosive atmospheres.
    (i) It is mandatory that the upper explosive limit (UEL) and lower 
explosive limit (LEL) for the fuel and/or fuel additive(s) that are 
being tested be determined. These limits can be found in the material 
safety data sheets (MSDS) for each substance and in various reference 
texts. The air concentration of the fuel or additive-base fuel mixture 
in the generation system, dilution/delivery system, and the exposure 
chamber system shall be calculated to ensure that explosive limits are 
not present.
    (ii) Storage, handling, and use of fuels or fuel/additive mixtures 
shall follow guidelines given in 29 CFR 1910.106.
    (iii) Monitoring for carbon monoxide (CO) levels is mandatory for 
combustion systems. CO shall be continuously monitored in the immediate 
area of the engine/vehicle system and in the exposure chamber(s).
    (iv) Air samples shall be taken quarterly in the immediate area of 
the vapor generation system and the exposure chamber system, or once at 
the beginning, middle, and end of the study for shorter studies. These 
samples shall be analyzed by methods described in paragraph 
(d)(6)(ii)(B) of this section.
    (v) With the presence of fuels and/or fuel additives, all electrical 
and electronic equipment must be grounded. Also, the dilution/delivery 
system and chamber exposure system must be grounded. Guidelines for 
grounding are given in 29 CFR 1910.304.
    (9) Quality control and quality assurance procedures--(i) Standard 
operating procedures (SOPs). SOPs for exposure operations, sampling 
instruments, animal handling, and analytical methods shall be written 
during the developmental phase of the study.
    (ii) Technicians/operators shall be trained in exposure operation, 
maintenance, and documentation, as appropriate, and their training shall 
be documented.
    (iii) Flow meters, sampling instruments, and balances used in the 
inhalation experiments shall be calibrated with standards during the 
developmental phase to determine their sensitivity, detection limits, 
and linearity.

[[Page 549]]

During the exposure period, instruments shall be checked for calibration 
and documented to ensure that each instrument still functions properly.
    (iv) The mean exposure concentration shall be within 10 percent of 
the target concentration on 90 percent or more of exposure days. The 
coefficient of variation shall be within 25 percent of target on 90 
percent or more of exposure days. For example, a manufacturer might 
determine a mean exposure concentration of its product's exposure 
emissions by identifying ``marker'' compound(s) typical of the emissions 
of the fuel or fuel/additive mixture under study as a surrogate for the 
total of individual compounds in those exposure emissions. The 
manufacturer would note any concentration changes in the level of the 
``marker'' compound(s) in the sample's daily emissions for biological 
testing.
    (v) The spatial variation of the chamber concentration shall be 10 
percent, or less. If a higher spatial variation is observed during the 
developmental phase, then air mixing in the chamber shall be increased. 
In any case, animals shall be rotated among the various cages in the 
exposure chamber(s) to insure each animal's uniform exposure during the 
study.
    (e) Data and reporting. Data shall be summarized in tabular form, 
showing for each 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.
    (1) Treatment of results. All observed results, quantitative and 
incidental, shall be evaluated by an appropriate statistical method. Any 
generally accepted statistical method may be used; the statistical 
methods shall be selected during the design of the study.
    (2) Evaluation of results. The findings of an inhalation 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 include the 
relationship between the concentration of the test atmosphere and the 
duration of exposure, and the severity of abnormalities, gross lesions, 
identified target organs, body weight changes, effects on mortality and 
any other general or specific toxic effects.
    (3) Test conditions. (i) The exposure apparatus shall be described, 
including:
    (A) The vehicle/engine design and type, the dynamometer, the cooling 
system, if any, the computer control system, and the dilution system for 
exhaust emission generation;
    (B) The evaporative emissions generator model, type, or design and 
its dilution system; and
    (C) Other test conditions, such as the source and quality of mixing 
air, fuel or fuel/additive mixture used, treatment of exhaust air, 
design of exposure chamber and the method of housing animals in a test 
chamber shall be described.
    (ii) The equipment for measuring temperature, humidity, particulate 
aerosol concentrations and size distribution, gas analyzers, fuel vapor 
concentrations, chamber distribution, and rise and fall time shall be 
described.
    (iii) Daily exposure results. The daily record shall document the 
date, the start and stop times of the exposure, number of samples taken 
during the day, daily concentrations determined, calibration of 
instruments, and problems encountered during the exposure. The daily 
exposure data shall be signed by the exposure operator and reviewed and 
signed by the exposure supervisor responsible for the study.
    (4) Exposure data shall be tabulated and presented with mean values 
and a measure of variability (e.g., standard deviation), and shall 
include:
    (i) Airflow rates through the inhalation equipment;
    (ii) Temperature and humidity of air;
    (iii) Chamber concentrations in the chamber breathing zone;
    (iv) Concentration of combustion exhaust gases in the chamber 
breathing zone;
    (v) Particle size distribution (e.g., mass median aerodynamic 
diameter and geometric standard deviation from the mean);
    (vi) Rise and fall time;
    (vii) Chamber concentrations during the non-exposure period; and
    (viii) Distribution of test substance in the chamber.

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    (5) Animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (i) Number of animals exposed;
    (ii) Number of animals showing signs of toxicity; and
    (iii) Number of animals dying.
    (f) References. For additional background information on this 
exposure guideline, the following references should be consulted.
    (1) Barr, E.B. (1988) Operational Limits for Temperature and Percent 
Oxygen During HM Nose-Only Exposures--Emergency Procedures [interoffice 
memorandum]. Albuquerque, NM: Lovelace Inhalation Toxicology Research 
Institute; May 13.
    (2) Barr, E.B.; Cheng, Y.S.; Mauderly, J.L. (1990) Determination of 
Oxygen Depletion in a Nose-Only Exposure Chamber. Presented at: 1990 
American Association for Aerosol Research; June; Philadelphia, PA: 
American Association for Aerosol Research; abstract no. P2e1.
    (3) Barrow, C.S. (1989) Generation and Characterization of Gases and 
Vapors. In: McClellan, R.O., Henderson, R.F. ed. Concepts in Inhalation 
Toxicology. New York, NY: Hemisphere Publishing Corp., 63-84.
    (4) Benedict, R.P. (1984) Fundamentals of Temperature, Pressure, and 
Flow Measurements. 3rd ed. New York, NY: John Wiley and Sons.
    (5) Cannon, W.C.; Blanton, E.F.; McDonald, K.E. The Flow-Past 
Chamber. (1983) An Improved Nose-Only Exposure System for Rodents. Am. 
Ind. Hyg. Assoc. J. 44: 923-928.
    (6) Chaddock, J.B. ed. (1985) Moisture and humidity. Measurement and 
Control in Science and Industry: Proceedings of the 1985 International 
Symposium on Moisture and Humidity; April 1985; Washington, D.C. 
Research Triangle Park, NC: Instrument Society of America.
    (7) Cheng, Y.S.; Barr, E.B.; Carpenter, R.L.; Benson, J.M.; Hobbs, 
C.H. (1989) Improvement of Aerosol Distribution in Whole-Body Inhalation 
Exposure Chambers. Inhal. Toxicol. 1: 153-166.
    (8) Cheng,Y.S.; Moss, O.R. (1989) Inhalation Exposure Systems. In: 
McClellan, R.O.; Henderson, R.F. ed. Concepts in Inhalation Toxicology. 
New York, NY: Hemisphere Publishing Corp., 19-62.
    (9) Cheng, Y.S.; Yeh, H.C.; Mauderly, J.L.; Mokler, B.V. (1984) 
Characterization of Diesel Exhaust in a Chronic Inhalation Study. Am. 
Ind. Hyg. Assoc. J. 45: 547-555.
    (10) Gillum, D.R. (1982) Industrial Pressure Measurement. Research 
Triangle Park, NC: Instrument Society of America.
    (11) Hinners, R.G.; Burkart, J.K.; Malanchuk, M. (1979) Animal 
Exposure Facility for Diesel Exhaust Studies.
    (12) Kittelson, D.B.; Dolan, D.F. (1979) Diesel exhaust aerosols. In 
Willeke, K. ed. Generation of Aerosols and Facilities for Exposure 
Experiments. Ann Arbor, MI: Ann Arbor Science Publishers Inc., 337-360.
    (13) Mokler, B.V.; Archibeque, F.A.; Beethe, R.L.; Kelly, C.P.J.; 
Lopez, J.A.; Mauderly, J.L.; Stafford, D.L. (1984) Diesel Exhaust 
Exposure System for Animal Studies. Fundamental and Applied Toxicology 
4: 270-277.
    (14) Moore, W.; et al. (1978) Preliminary finding on the Deposition 
and Retention of Automotive Diesel Particulate in Rat Lungs. Proc. of 
Annual Meeting of the Air Pollution Control Assn, 3, paper 78-33.7.
    (15) Raabe, O.G., Bennick, J.E., Light, M.E., Hobbs, C.H., Thomas, 
R.L., Tillery, M.I. (1973) An Improved Apparatus for Acute Inhalation 
Exposure of Rodents to Radioactive Aerosols. Toxicol & Applied 
Pharmaco.; 1973; 26: 264-273.
    (16) Rao, G.N. (1986) Significance of Environmental Factors on the 
Test System. In: Hoover, B.K.; Baldwin, J.K.; Uelner, A.F.; Whitmire, 
C.E.; Davies, C.L.; Bristol, D.W. ed. Managing conduct and data quality 
of toxicology studies. Raleigh, NC: Princeton Scientific Publishing Co., 
Inc.: 173-185.
    (17) Spitzer, D.W. (1984) Industrial Flow Measurement. Research 
Triangle Park, NC: Instrument Society of America.
    (18) 40 CFR part 798, Health effects testing guidelines.
    (19) 29 CFR part 1910, Occupational safety and health standards for 
general industry.

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    (20) Federal Register, 42 FR 26748, May 25, 1977.

[59 FR 33093, June 27, 1994, as amended at 61 FR 58746, Nov. 18, 1996; 
61 FR 36512, July 11, 1996]