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OAR Box 1827 

Prepped by Charmelle Mathews 

Document Number: 

138) II-A-8 

Docket Number: 

A-90-07 

Printed 11/19/2008 12:40:48 PM Sheet 138 of 147 
Ii — %. 

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General Health Testing Guideline for Inhalation 

Exposure to Fuels and Fuel Additives 

Final Report 

Prepared by: 

Yung-Sung Cheng and Edward B. Barr 

under 

EPA Contract No. 68-C8-0024 

Master Work Assignment No. 2-1, Subtask 2 

Prepared for: 

Office of Mobile Sources 

U.S. Environmental Protection Agency 

September 30, 1991 
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(a) Purpose. In the assessment and evaluation of potential health effects of emissions of 

motor vehicle engines operated on different types of fuels and fuel additives (F/FA), 

determination of inhalation toxicity is required in § 211 of the Clean Air Act. Laboratory 

studies involve the exposure of animals by inhalation to the exhaust and (separately) to the 

evaporative emissions produced by the motor vehicle fuels and fuel additives. 

The standard test guidelines and protocols for acute, subacute (or subchronic) and chronic 

inhalation experiments as described in the Health Effects Testing Guidelines (40CFR 798) are 

useful for conducting exposures of single chemical compounds or simple mixtures of several 

compounds. However, these testing guidelines do not provide sufficient technical direction for 

conducting animal exposures to fuel emissions, because of the following two reasons. First, the 

combustion exhaust from motor vehicle engines requires complex generation methods with 

specialized equipment including an engine, test stand and dynamometer, engine control system, 

cooling system, and exhaust dilution system. Secondly, the heated engine exhaust is a 

multiphasic complex mixture of soot particles and gases. The soot particles have a carbon core 

with hundreds of condensed organic compounds of fuel and incomplete combustion products 

(Cheng, et al., 1984). The exhaust gases include carbon monoxide, carbon dioxide, nitrogen 

oxides, sulfur dioxide, and hydrocarbons. Special sampling and analytical methods are required 

to characterize the exposure atmospheres, especially for the complex mixture of the engine 

exhaust. 

The purpose of this report is, therefore, to provide additional information on 

methodologies required to conduct inhalation exposures of combustion and evaporative emissions 

from automotive engines using F/FA. 

(b) Definitions. (1) An acute inhalation study is 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. Several concentration levels and a concurrent untreated group shall be used. 

(2) The subacute (or subchronic) inhalation study is a repeated exposure by inhalation 

for part (approximately 10 %) of a life span. A subacute inhalation study may be carried on 

after initial information on toxicity has been obtained by an acute inhalation study. 

(3) A chronic inhalation study is a prolonged and repeated exposure by inhalation for 

the life span of the animal. 

(4) The aerodynamic diameter applies to the size of aerosol particles. It is 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 due to sedimentation and impaction 

mechanisms. 

(5) The diffusion diameter also applies to the size of aerosol particles. It is the diameter 

of a sphere that has the same diffusion coefficient 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 due to Brownian diffusion. For spherical 

particles of unit density, the aerodynamic diameter and diffusion diameter are the same as the 

geometric diameter. However, for an aggregate aerosol, such as soot particles from engine 

exhaust, the aerodynamic diameter and diffusion diameter are different. For micrometer-size 

particles, the aerodynamic diameter is usually sufficient to predict respiratory tract deposition. 

For soot particles, the diffusion diameter is often measured because the particles are 
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submicrometer size, and diffusion deposition dominates respiratory deposition. 

(6) The mass median diameter is the calculated aerodynamic diameter (MMAD) or 

diffusion diameter (MMDD), 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 50% will be smaller than the median diameter. The mass median diameter and the 

geometric standard deviation describe the particle-size distribution. 

(7) Concentration refers to an exposure level. Exposure is expressed as weight or 

volume of test substance per volume of air (mg/m3 for aerosols) or as parts per million (ppm) 

for vapors and gases. 

(8) Data acquisition is the collection of signals from a transducer or from a system of 

transducers. 

(9) A dynamometer is a mechanical device used to measure power. 

(10) The fuels used in spark-ignition engines and compression-ignition engines of 

automotive vehicles include gasoline, compression-ignition fuel, compressed or liquified natural 

gas (CNG or LNG, mainly methane and propane), ethanol, and methanol. Fuel additives 

blended into gasoline include ethanol, methyl tertiary butyl ether (MTBE) and tertiary amyl 

methyl ether (TAME). 

(11) Material safety data sheet (MSDS) contains information on the physical, chemical, 

and hazardous characteristics of a given chemical. 

(12) Reynolds number is a dimensionless number that is proportional to the ratio of 

inertial forces to factional forces acting on a fluid. It quantitatively provides a measure of 

whether flow is laminar or turbulent. Flow through pipes is laminar for a Reynolds number less 

than 2000, and turbulent for a Reynolds number greater than 4000. 

(13) A transducer is a device that measures a physical phenomena. It is actuated by 

electrical power and transforms a physical quantity into an electrical signal. 

(c) Principles and design criteria of inhalation exposure systems. Proper conduct of 

inhalation toxicity studies of fuel and fuel-additive emissions 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) The generation of fuel and fuel-additive exposure atmospheres may be categorized 

into two major types of systems: diluted, whole-combustion emissions of motor vehicle engines, 

operated on different types of fuel and/or fuel additives, and evaporative emissions from 

different types of fuel and/or fuel additives. 

(i) Emissions from combustion of motor vehicle engines are generated either by the test 

engine mounted on a dynamometer base plate or a complete vehicle operated on a chassis 

dynamometer. Critical to either system is control of the engine operating conditions to 

accurately simulate driving conditions as prescribed by the given exposure study. Normally 

operation of the engine system is dependent on a computer-based control system, but it may in 

some circumstances be manually controlled. For example, if the study requires the engine to 

operate at a steady-state speed (rpm) or load, the fuel injection system or carburetor may be 

manually set to give the desired operating conditions. If, however, the engine is required to 

follow a driving schedule (40CFR 86), a computer-controlled system is strongly recommended. 
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(A) There are four major types of motor vehicle engines used in fuel and/or fuel additive 

studies: a light-duty, spark-ignition (Otto cycle) engine, a light-duty, compression-ignition 

engine, a heavy-duty, spark-ignition engine and a heavy-duty, compression-ignition engine. 

Engine classification will be in accordance with provisions as described in 40CFR 86. The 

engine will be linked to either automatic or manual transmission. For studies that follow a 

driving schedule, an automatic transmission is recommended. 

(B) The dynamometer is used to simulate road load power. Usually the dynamometer 

is linked to a fly wheel that is used to simulate the inertial component (weight) of the test 

vehicle. Absorption dynamometers are normally the type used in engine test systems. This type 

absorbs the power from the test engine and dissipates it as heat. There are basically four kinds 

of absorption dynamometers: Prony brake, fan brake, water brake, and eddy-current brake. Any 

of these is acceptable for use with the engine test system, provided that accurate measurement 

of shaft speed and torque can be obtained. Guidelines for dynamometer accuracy are detailed 

in 40CFR §86.1308-84. These guidelines are particularly important when the engine system is 

operated in accordance with a defined driving schedule. 40CFR 86 provides guidelines for 

acceptable operating ranges of torque and engine (shaft) speeds. 

(C) There are five different driving schedules that have been widely used for light-duty 

engines and vehicles; New York City cycle, Federal Test Procedure urban schedule (40CFR 86, 

Appendix I), Hi way fuel economy test, 7-mode cycle (Moore, 1978), and the Federal short cycle 

(Federal Register. 1977). Steady-state driving schedules, where the engine is under constant 

load and constant speed, have also been widely used. Driving schedules for heavy-duty engines 

and vehicles are given in 40CFR 86, subparts H, I, J, and N. The driving schedule used for 

the emission inhalation exposure will be based on the specific requirements for the given study. 

(D) Well-defined operation of the engine system is critical to ensure that engine 

emissions simulate emissions from vehicles operating under actual road conditions. At a 

minimum the shaft speed and torque shall be monitored during operation of the engine system 

and shall meet the specifications as detailed in 40CFR §86.1308-84. It is strongly recommended 

that other critical parameters be monitored to ensure proper operation of the engine system. 

These are the oil pressure, oil temperature, engine coolant temperature, and dynamometer 

coolant temperature. Frequency of monitoring and recording these parameters will be specified 

in the given exposure study. 

(E) If the engine system is following a driving schedule, control is most likely 

accomplished via a computer-based, real-time control system. The major components of such 

a system are the transducers (sensors), the engine and dynamometer data acquisition hardware, 

the acquisition software, and the computer. 

(1) Transducers are the devices which measure temperature, pressure, rpm, or other 

physical parameters. Transducers convert the measured parameters into electronic signals 

(resistance, current, voltage, frequency) that can be interpreted by the data acquisition hardware. 

The wide range of transducer types, manufacturers, and cost makes it beyond the scope of this 

report to cover them in any detail. In-depth discussions of transducers are found in other 

sources (Sheingold, 1981; Tucker, 1990; Klier, 1990). General guidelines for selection of 

transducers are as follows. The transducer must be capable of functioning in the engine 

environment. Transducers will be exposed to high temperatures (250-30O°F), corrosive liquids, 

fumes and vapors, and mechanical vibration. Transducers must provide reproducible and 

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accurate results in the range of interest for the specific parameter being measured, and they must 

be stable over time and use. Temperatures for engine and dynamometer coolants will have an 

upper range of about 250-300°F. Oil pressure measurements will be up to 50 psig, or higher. 

Engine and dynamometer rpm will be range of 50 to 5000. The transducer must be compatible 

with the data acquisition hardware. 

(2) Data acquisition hardware is the interface between the transducers and the computer. 

This hardware must convert the transducer output into a form usable by the computer. As with 

transducers, there is a wide range of commercially available hardware. Selection of the 

hardware will be based on the requirements of the study. 

(3) Data acquisition software converts raw information from the data acquisition 

hardware into formatted data in a defined scale and units. Selection of the software will be 

based on the requirements of the study and the computer system. 

(4) The computer runs the data acquisition software, stores the results of the data 

acquisition hardware, and performs analysis and data reduction of the results. Selection of the 

computer system is based on the requirements of the study. 

(F) Maintenance of the engine system should be performed in a manner consistent with 

that expected for an operational vehicle. It is recommended that scheduled maintenance on the 

engine system be performed as specified in the manufacturer's maintenance instructions which 

are furnished with the commercial vehicle. 40CFR §86.090-25 provides detailed guidelines on 

engine/vehicle maintenance. 

(G) The exhaust system of a motor vehicle is incorporated into the chassis design. For 

test systems that have the engine mounted on a test stand, a chassis-type exhaust system must 

be installed which is comparable to the exhaust system of the vehicle for which the engine was 

designed. 40CFR §86.1308-84 provides information on the selection and design of the exhaust 

system. 

(H) During long-term (non-acute) exposure studies, it is critical that the engine system 

be operational throughout the study. Major repair/maintenance of the engine system or failure 

of the system can disrupt the exposure and may have a severe impact on the study. A reserve 

or back-up engine system must be included in the design of the exposure system. This will 

provide a capability to switch the engine system to the reserve unit in order to maintain 

continuity of the exposure. 

(ii) Evaporative emissions. Various methods for the generation of gases and vapors have 

been described in the literature (Barrow, 1989). Generation of fuel and/or fuel additive vapors 

may include any method which does not alter the chemical form of the fuel/fuel additive or 

generate other vapors and/or particulates. Vapor concentration is commonly expressed as a 

volume-to-volume measurement. The volumetric fraction of the fuel/fuel additive in exposure 

air is usually expressed as percent or ppm. The two primary parameters affecting vapor 

concentration are partial pressure of the fuel/fuel additive(s), and temperature. Maximum 

concentration of evaporative emissions (MCEE) will be a function of the vapor pressure of the 

fuel/fuel additive(s). 

MCEE (ppm1=Fuel vapor pressure(mm Hp at temperature. T, x(lxl06). 

Chamber pressure (mm Hg at temperature, T) 
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During long-term (non-acute) exposure studies, it is critical that the vapor generation system be 

operational throughout the study. Failure of the vapor generation system can disrupt the 

exposure and may have a severe impact on the study. A reserve or back-up generation system 

must be included in the design of the exposure system. This will provide a capability to switch 

the generation system to the back-up system in order to maintain continuity of the exposure. 

(2) Dilution and delivery systems. The dilution of emissions produced by the generation 

system serves three purposes. Primarily, dilution simulates the conditions of exhaust emissions 

released to the atmosphere from operational motor vehicles. Studies have shown that under 

roadway conditions compression-ignition exhaust is diluted more than 1000 to 1 in less than 1 

second (Kittelson and Dolan, 1979). Spark-ignition exhaust dilution rates are expected to be of 

the same order. Formation of particle species is strongly dependent on the dilution rate. The 

dilution system must be capable of rapid dilution of engine emissions in order that the resulting 

formation of particle species simulates the same process under roadway conditions. Dilution also 

provides control of the emissions concentration delivered to the exposure system. The dilution 

system serves 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. Such responses may mask the actual health effects caused 

by the emissions or may mislead the investigators to misinterpret results. 

(i) Integral to the dilution system is the delivery system. 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) Flow requirements. Dilution of combustion emissions must be carefully considered. 

The hot emissions from an engine or vehicle must be diluted and cooled sufficiently to simulate 

as closely as possible the conditions of the vehicle under roadway conditions. Formation of 

compounds, particulates, and gases ideally will be identical to those under actual roadway 

conditions. Exhaust emissions should be diluted with clean filtered air at a flow rate in the 

range of 8:1 or higher (Hinners, et al., 1979). The dilution section should be of such 

dimensions as to provide residence times for the emissions in the order of < 1 second to several 

seconds before the emissions are introduced to the chamber system. This will allow for the 

formation of emissions to simulate actual road conditions. As a result of the high flow rates in 

the emission dilution section, it may be necessary to extract a fraction of the diluted emissions 

for delivery to the chamber system and additional dilution. The remaining emissions are 

normally exhausted to the atmosphere outside of the exposure facility. First-stage dilution of 

the engine exhaust system will determine the nature of the emissions formed; therefore, it is a 

major consideration in dilution system design. Dilution ratio of air to exhaust emissions should 

be a minimum of 8:1 to minimize particle loss and to prevent condensation of the emissions 

(Hinners, et al.,1979). Dimensions of the first-stage-dilution section should be such that, at a 

minimum, the flow Reynolds number is 70,000 or greater (Mokler, et al., 1984). This will 

maintain highly turbulent flow conditions so that there is complete mixing of the exhaust 

emissions. The engine exhaust system should connect to the-first-stage-dilution section at 90° 

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. 

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(iii) Materials. A major consideration in the design of the dilution and delivery systems 

is to ensure that the emissions are not chemically or physically altered. In designing the dilution 

and delivery systems, the use of plastic, PVC, or similar pipe and tubing should be avoided. 

Such materials may be chemically reactive with the emissions. Also, particulate emissions will 

become highly charged if non-conducting tubing is used. Metal tubing and pipe are 

recommended for all dilution and delivery systems. Copper, brass, and aluminum pipe and 

tubing should be avoided if there exists a possibility of chemical reaction occurring between 

emissions and tubing. Stainless steel pipe and tubing are recommended as the best choices. 

(iv) Wall losses. The delivery system should 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. Flow reynolds number of 1000-3000 will ensure minimal wall losses. Flow 

rate and tubing diameter will determine the flow Reynolds number. Also the length of the 

delivery lines to the exposure chamber system should be minimized. The number and degree 

of bends in the delivery lines also should be minimized. Wall losses of 50% or greater are not 

unusual; therefore, it is important to minimize losses. 

Calculation of delivery system losses is not practical for complex systems such as used 

with most exposure systems. Measurement of system losses is not recommended. 

(v) Delivery systems differ significantly for whole-body exposure chambers vs. nOseonly exposure systems. Flow rates through whole-body chamber systems are of the order of 50 

liters per minute to 500 liters per minute. Nose-only systems are of the order of less than 50 

liters per minute. To maintain laminar flow conditions, the tubing size will be different for each 

system; however, the principles described in section (iv) apply to both systems. 

(3) Exposure chamber systems. There are two basic types of dynamic inhalation exposure 

chambers: whole-body chambers and nose-/head-only exposure chambers. (Cheng and Moss, 

1989). In these dynamic chambers, the test atmosphere is being continuously delivered to and 

exhausted from the chamber in a flow-through manner. The flow rate through a chamber should 

be maintained at 10 to 15 air changes per hour. 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. The exposure chamber should be designed in such a way as to provide uniform 

distribution of exposure concentrations in all compartments (Cheng et al., 1989). 

(i) In a whole-body exposure chamber, animals are housed in separate compartments 

inside the chamber, where the whole surface area of an animal is exposed to the test material. 

Whole-body exposure chambers usually have provisions for food and water, so that the animals 

can be housed in the chamber during the whole exposure period, or in cages that can be wheeled 

in or out the chamber. These chambers have a large volume to accommodate large number of 

animals. The spaces required for different animal species should follow the guidelines published 

by the U.S. Department of Health and Human Services (1985). In general, the volume of 

animal bodies occupy less than 5% of the chamber volume. Animals are under little stress in 

these chambers; therefore, the whole-body exposure chambers are suitable for subchronic or 

chronic exposure or experiments that required long exposure hours. 

(ii) In head/nose-only exposure chambers, only the head portion (oral-nasal) of the 

animal is exposed to the test material to minimize skin contamination. The chamber volume 

and flow rates are much less than in the whole-body exposure chambers; therefore, only a 

minimum amount of test material is used. Animals are housed in separate boxes or tubes, where 
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the animal's breathing can be easily monitored. Head/nose-only exposure chambers are used 

frequently for exposures of dogs, monkeys and humans. Exposure chambers with multiple ports 

to accommodate up to 90 rodents are available (Raabe et al., 1973; Cannon et al., 1983). 

Rodents are usually retrained in a tube holder with a much-reduced heat capacity. It is laborintensive to operate this type of exposure chambers. Therefore, the head/nose-only exposure 

chamber is suitable for short-term exposures or when use of a small amount of test material is 

required. 

(d) Inhalation exposure procedures. The proper conduct of an inhalation study will 

require that procedures for the selection of the test animal, handling and care of the test animal, 

establishment of the proper environmental conditions in the exposure chamber, monitoring of 

the exposure emissions or vapors, monitoring of the environmental conditions in the exposure 

system, and safety issues are well defined. 

(1) Animal selection for inhalation exposure procedures, (i) A mammalian species shall 

be used for testing. A variety of rodent species may be used, although the rat is the preferred 

species. Commonly used laboratory strains shall be used. 

(ii) Young adult animals shall be used. At the commencement of the study the weight 

variation of animals shall not exceed ± 20 percent of the mean weight for each sex. The 

animals should be randomly assigned to each group according the weight. 

(iii) An equal number of male and female animals shall be used in each concentration 

level. Females shall be nulliparous and nonpregnant. 

(iv) The number of animals used in each concentration level and control group depend 

on the type of study, number of biological end points used in the toxicity evaluation, and the 

animal species. For an acute study, at least five animals of each sex shall be used in each test 

group. For a subchronic study, at least 10 rodents of each sex shall be used in each test group. 

If another mammalian species is selected (e.g., dog, rabbit, or non-human primate), at least 

eight animals (four males and four females) shall be used. For a chronic study, at least 20 male 

and 20 female rodents shall be used in each test group. If another mammalian species is 

selected, at least eight animals ( four males and four females) shall be used. 

(A) If interim sacrifices are planned, the number of animals shall be increased by the 

number of animals 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 an untreated or sham 

treated control group. Except for treatment with the test substance, animals in the control group 

shall be handled in a manner identical to the test group animals. These animals shall be housed 

in similar exposure chambers and exposed to filter supply air. 

(2) Animal handling and care. A key element in the conduct of inhalation exposure 

studies is the proper handling and care of the test animals. Guide for the Care and Use of 

Laboratory Animals (U.S. Department of Health and Human Services, 1985) provides guidelines 

for animal care and use. Topics covered by this document include the following: housing, food 

and water, quarantine, disease control and treatment, anesthesia and euthanasia, ventilation, 

lighting, and noise control. The Guide also provides recommendations on housing space 

requirements for various species. The responses of animals to inhaled exhaust emissions can 

be modified by the prevailing conditions of housing and care before, during, and after an 

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exposure period; therefore, it is strongly recommended that the exposure conditions conform 

strictly with the conditions for housing set forth in the Guide. 

(i) Exposure systems using head/nose-only exposure chambers require no special daily 

chamber maintenance. Chambers should be inspected daily 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. Chambers will be cleaned and maintained in accordance 

to guidelines set forth in Guide for the Care and Use of Laboratory Animals (U.S. Department 

of Health and Human Services, 1985). 

(ii) Food and water will be withheld from animals for head/nose-only exposure systems. 

For whole-body-exposure systems, food and water will be available ad libitum at all times when 

the generation system is not operating. During operation of the generation system, food will be 

withheld to avoid possible contamination by emissions. Ingestion of contaminated food may 

affect animal response to the emissions. Water must be provided at all times. 

(iii) In whole-body-exposure chambers, animals shall be housed in individual caging. 

The minimum cage size per animal will be in accordance to guidelines set forth in Guide for the 

Care and Use of Laboratory Animals (U.S. Department of Health and Human Services, 1985). 

(3) Concentration levels and concentration selection, (i) In acute toxicity tests, three 

exposure concentrations and a control group should be used and spaced appropriately to produce 

test groups with a range of toxic effects and mortality rates. The data should 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, it is desirable to have a concentrationresponse relationship as well as a no-observed-toxic-effect level. Therefore, at least three 

concentration levels with a control shall be used. Concentrations shall be spaced appropriately 

to produce test groups with a range of toxic effects. The criteria for selecting concentration 

level have been published (40CFR §§798.2450 and 798.3260). 

(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 should be used to dilute the exhaust emissions during 

exposure and during non-exposure periods to maintain environmental conditions that are free of 

trace gases, dusts, and microorganisms on the test animals. Ten to fifteen (chamber volume) 

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 (Cheng and Moss, 

1989). 

(ii) Temperature is one of the most important environmental parameters. Recommended 

ranges of temperature for various species are given in Guide for the Care and Use of Laboratory 

Animals (U.S. Department of Health and Human Services, 1985). The recommended 

temperature ranges will be used for establishing temperature conditions of whole-body-exposure 
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chambers. In head/nose-only chambers, animals are usually confined inside exposure tubes with 

little or no air circulating around the body. The exposure tube prevents heat transfer by 

evaporation and convection, leaving radiation heat transfer from tube walls as the only effective 

means of heat removal. Thermoregulation in rodents is primarily by the tail. Modifications of 

the exposure tube can be used to expose the tail outside the tube to allow the animal to better 

regulate body temperature (Cheng and Moss, 1989). 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 (Barr, 1988). Maximum 

temperature for rats and mice in head/nose-only tubes is 23°C. 

(iii) Relative humidity. The relative humidity in the chamber air is important for heat 

balance. The relative humidity will affect feed consumption, behavior, disease, and chemical 

effects on test animals (Rao, 1986). The recommended relative humidity range for rodents in 

exposure chambers is 30%-70% (Guide for the Care and Use of Laboratory Animals (U.S. 

Department of Health and Human Services, 1985)). 

(iv) Lighting. Lighting is the most important environmental factor for rodents. Because 

rodents are nocturnal animals, light will influence the circadian rhythm, endocrine functions and 

reproductive processes. Due to its profound effect on the endocrine system, light will have a 

marked effect on the animal's physiology. As a result, this can significantly affect the 

toxicologic responses of rodents to exhaust emissions. Light intensity of 30 foot candles at 3 

ft. from the floor of the exposure facility is recommended (Rao, 1986). 

(v) Noise. The auditory system in test animals is highly developed and, therefore, is 

sensitive to environmental noise. Elevated noise levels, especially noise levels associated with 

engine or vehicle systems, can result in toxicological effects on the test animals. 

(vi) Oxygen content. Sufficient oxygen must be available to the test animals in the 

exposure chamber. This is especially important in head/nose-only chambers that have small 

volume to animal ratios (less than 1000 cc/animal). 

(5) Exposure Conditions. For subchronic and chronic studies, the animals should be 

exposed to the test substance at the minimum of 5 days/week, 6 hours/day. This exposure 

regime is to simulate occupational exposures. Continuous exposure for at least 16 hours per day 

and 7 days per week is used to simulate exposure of general population to engine emissions. 

(6) In some special cases, discontinuous or peak exposures will be used to simulate 

unsteady concentration profiles of evaporative emissions. The vapor concentration during the 

exposure period will be increased from low-level (or zero) stable concentrations 

to a peak concentration, then return to the stable concentration for a defined period of time. In 

these cases, a real-time monitor is needed to ensure that the concentration profile is within the 

specified limits. 

(7) The exposure atmosphere must be monitored to ensure that exposure levels are at the 

target values or within stated limits. 

(i) 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) Exposure to combustion exhaust is often conducted with the engine operated at a 

driving cycle, such as the Federal Test Procedure, to simulate urban driving conditions. The 

exhaust aerosol concentration has been shown to vary as a function of time (Cheng et al., 1984). 

Therefore, integrated samples of aerosol concentration should be taken daily during the 

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exposure from a single representative sample port in the chamber near the breathing zone of the 

animals. Gas samples should be taken frequently (no less than once a week) to determine 

concentrations (ppm) of individual components including CO, C02, NOx, S02, and fuel vapors. 

The gas sample should be taken in one or several complete driving cycles. 

(B) At a minimum, vapor concentrations of the evaporative emissions in the chamber 

should be determined hourly. The sample should be taken from a single, representative 

sampling port. 

(C) Distribution of aerosol or vapor concentration in exposure chambers should be 

determined without animals during the developmental phase of the study, and with animals 

during the exposure to ensure that animals in different locations of the chamber receive a similar 

exposure atmosphere. For head/nose-only exposure chambers, it may not be possible to monitor 

the chamber distribution during the exposure, because the exposure ports contain animals. 

(D) The particle size distribution of the combustion exhaust aerosol should be determined 

monthly. 

(E) Chamber rise and fall times. The time required for the exposure concentration to 

reach 90% (T90) of the stable concentration after the generator is turned on, and the fall time 

when the chamber concentration decreases to 10% of the stable concentration after the generation 

system is stopped should be determined in the developmental phase of the study. Timeintegrated samples collected for calculating exposure concentrations should be taken after the rise 

time. The daily exposure time is the exposure time plus T90, e.g., 6 hours +T90. 

(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 combustion exhaust studies, time-integrated samples using a filter as a collection 

substrate is recommended. Gravimetric determination of the aerosol concentration (mg/m3) 

should be made. Other aerosol instruments, such as photometers, may be used to determine the 

stability of aerosol concentration in the chamber. Combustion gases should 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. 

(B) For evaporative emission studies, concentration of fuel vapors can usually be 

determined by using a gas chromatograph (GC) and/or infrared spectrometry (IR). Grab samples 

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. 

(C) Chamber distribution. Spatial variation of chamber concentrations can be calculated 

by simultaneously sampling different locations in an exposure chamber. The coefficient of 

variation of samples from all locations is then an estimate of the spatial variation. If an on-line, 

real-time monitor is available, an alternative method of sampling from a reference point and 

other locations in.series can also be used (Cheng et al., 1989). 

(D) Particle size distribution. Because the size range of combustion aerosols is from 0.1 

to several micrometers, the aerosol instruments used should have the corresponding range. 

These include a cascade impactor with appropriate size cuts, a low-pressure impactor, or a 

11 
P.13 

combination of a cascade impactor and a parallel diffusion battery. 

(E) The rise and fall times of the chamber concentration should be taken by continuously 

monitoring the test material concentration in a chamber after the generator is turned on or after 

the generator is stopped. Real-time monitors, such as photometers for aerosols, and GC or IR 

detectors for vapors, should be used. 

(8) Monitoring chamber environmental conditions. Monitoring of chamber environmental 

parameters (air flow, temperature, relative humidity, lighting, noise levels, and oxygen content) 

is necessary to ensure that chamber conditions are within prescribed guidelines for the proper 

maintenance of the test animals. Monitoring may be performed by a computer system or 

exposure system operating personnel. 

(i) 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 (Benedict, 1984; Spitzer, 1984). 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% of full scale range. For 

some devices, such as orifice meters and venturi meters, flow is calculated based on the pressure 

differential across the flow device. Pressure measurement may be accomplished using 

manometers, electronic pressure transducers, magnehelics, or similar devices (Gillum, 1982). 

Accuracy of the pressure device must be + 5% of full scale range. Pressure measurements 

must be continuous and recorded at least every 30 minutes. 

(ii) Temperature may be measured using thermometers, RTD's, thermocouple, 

thermistors, or other devices (Benedict, 1984). The temperature of exposure chambers must be 

monitored continuously and recorded at least every 30 minutes. Because rodents are very 

temperature sensitive, particularly at higher temperatures, 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°C (80°F). The exposure system must 

be discontinued and emergency procedures enacted to immediately reduce temperatures or 

remove test animals from high temperature environment when chamber temperatures exceed 

29°C. Accuracy of the temperature monitoring device will be ± 1 °C for the temperature range 

of 20-30°C. 

(iii) Relative humidity may be measured using various devices (Chaddock, 1985). 

Selection of the measuring device must be done carefully. Many of the commercially available 

sensors are not suitable for use in animal chambers. The exposure atmosphere or the excreta 

from the animals may affect the sensitivity or calibration of the sensor. Also care must be taken 

that the sensors are not affected when the chamber is serviced by animal care personnel. The 

relative humidity of exposure chambers must be monitored continuously and recorded at least 

every 30 minutes. 

(iv) Lighting shall be measured quarterly. 

(v) Noise level in the exposure chamber(s) shall be measured quarterly. 

(vi) Oxygen content is critical in nose-only chamber systems where the chamber volume 

per animal is of the order of 200-1000 cc per animal. If air flow is interrupted to the chamber, 

animals can suffocate within several minutes (Barr, et al., 1990). An oxygen sensor shall be 

located at a single position in the chamber and a lower alarm limit of 18% shall be used to 

12 
P.14 

activate an alarm system. 

(9) Safety procedures and requirements. Safety is the primary consideration in the design 

and operation of an inhalation exposure system. Unique problems are associated with the 

operation of combustion and fuel/fuel-additive vapor generation systems. 

(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 are listed 

in the Material safety data sheets (MSDS) for each substance. The air concentration of the 

fuel/fuel additive 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 fuel/fuel additives shall follow guidelines given in 

Occupational Safety and Health Standards for General Industry (29CFR §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 area. 

(iv) Air samples shall be taken quarterly in the immediate area of the vapor generation 

system and the exposure chamber system. These samples shall be analyzed by methods 

described in section (d)(7)((ii)(B). 

(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 Occupational Safety and Health 

Standards for General Industry (29CFR §1910.304). 

(10) Quality control and quality assurance procedures, (i) Standard operation procedures 

(SOP). SOPs for exposure operations, sampling instruments, animal handling, and analytical 

methods should be written during the developmental phase. 

(ii) Training of exposure operators should be performed and documented. 

(iii) Flow meters, sampling instruments, and balances used in the inhalation experiments 

shall be calibrated with standards during the developmental phase to determine the sensitivity, 

detection limits, and linearity. During the exposure period, instruments should be checked for 

calibration and documented to ensure that each instrument still functions properly. 

(iv) The daily mean exposure concentration shall be within 15% of the target 

concentration for no less than 90% of total exposure days. The overall mean exposure 

concentration during a given study shall be within 10% of target concentration. The coefficient 

of variation shall be within 25% for no less than 90% of total exposure days. The overall 

coefficient of variation shall within 25% during a given study. 

(v) Excursions. In the event that the daily mean exposure concentration is greater than 

±15% of target concentration, procedures shall be defined to adjust the generation system 

and/or dilution/delivery system to re-establish exposure concentrations within limits defined in 

section (d)(10)(iv). Such excursions shall be documented and actions taken to rectify excursions 

shall also be documented. 

(vi) Chamber distribution. The spatial variation of the chamber concentration should be 

smaller than 8%. If a higher spatial variation is observed during the developmental phase, 

animals shall be rotated, or air mixing in the chamber shall be increased. 

(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 

13 
P.15 

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 should 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 substance and the duration of exposure, and 

the severity, of abnormalities, gross lesion, identifies 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 engine design and type, the dynamometer, the cooling system, the computer 

control system, and the dilution system, for engine exhaust emissions, or 

(B) the generator type, the generator design, and the dilution system, for evaporative 

emissions. 

Other test conditions such as the source of air, fuel type, 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 shall be recorded. The daily record should document the 

date, the starting and stop times of the exposure, number of samples taken during the day, daily 

concentrations, 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 (median diameter and geometric standard deviation). 

(vi) Rise and fall time. 

(vii) Chamber concentration during the non-exposure period. 

(viii) Distribution of test substance in the chamber. 

(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. 

(iii) Number of animals dying. 

(f) Cost estimates for the engine exhaust, generation, and delivery system shown in 

Table 1 are not intended to be all-inclusive or absolute. The cost estimates provide broad 

guidelines for major cost items. Costs excluded from estimates are site preparation and 

installation of test stands, installation of exhaust dilution and delivery systems, and expendable 

14 
P.16 

supplies. 

(1) The engine test stand and dynamometer system. The engine test stand includes 

engine (light duty compression-ignition, heavy duty compression-ignition, light duty internalspark ignition, or heavy duty internal-spark ignition), the engine mounting unit, and the external 

engine cooling system. The dynamometer includes eddy-current (or equivalent) dynamometer, 

engine linkage unit, fly-wheel and housing, and the dynamometer controller. 

(2) The engine control system includes data acquisition system, computer, control 

software, and fuel injection/carburetor control system. Data acquisition system provides 

interface between the engine and dynamometer systems and the computer control software. It 

is assumed that the engine control system will be computer-based. In specific systems, the 

engine may be controlled manually or by some electro-mechanical system. In this case, costs 

will not apply. In a computer-based control system, the computer can range from a IBM-PC 

or compatible to a mainframe computer. The cost of the computer is therefore variable and thus 

is not included. The software is computer-specific and is therefore also variable. A fuel control 

system is required in a computer-based system and is normally controlled by low-voltage signal 

(mV to 10 V range) or a 4-20 mA loop. Engine control systems range from systems designed 

and constructed in-house to turn-key systems. Cost estimates in Table 1 assume a modular 

system constructed of hardware and software available from commercial sources. 

(3) Exhaust dilution and distribution system. The exhaust generated by the engine 

system must be cooled, diluted, and delivered to the animal exposure system. Dilution is 

normally a minimum of 8:1 (air:exhaust). 

15 
P.17 

Device/System Description Estimated Cost 

Engine Test Stand and Dynamometer 

Engine 

Engine Cooling System 

Dynamometer system (includes inertia 

stand, flywheel, and controller) 

compression-ignition 

(light duty): $ 8,000 

compression-ignition 

(heavy duty): $22,000 

spark-ignition 

(light-duty): $ 5,000 

spark-ignition 

heavy-duty): $15,000 

$3,000 

$40,000 

Engine Control System 

Data Acquisition System 

Computer [PC-AT or equivalent 

Control Software 

Fuel Injection/Carburetor Control System 

$ 3,000 

$3,000 

$ 4,000 

$ 2,000 

Exhaust Dilution and Distribution System 

Main exhaust Dilution System 

Diluted Exhaust Distribution System 

Total Estimated Cost 

$ 7,000 

$ 4,000 

$71,000-98,000 

Table 1: Cost estimates for combustion emissions exposure system 

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P.18 

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