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 ------- |